Lettuce cultivar regency 3.0

ABSTRACT

A lettuce cultivar, designated Regency 3.0, is disclosed. The invention relates to the seeds, plants and plant parts of lettuce cultivar Regency 3.0 and to methods for producing a lettuce plant by crossing the cultivar Regency 3.0 with itself or another lettuce cultivar. The invention further relates to methods for producing a lettuce plant containing in its genetic material one or more transgenes and to the transgenic lettuce plants and plant parts produced by those methods. This invention also relates to lettuce cultivars or breeding cultivars and plant parts derived from lettuce cultivar Regency 3.0, to methods for producing other lettuce cultivars, lines or plant parts derived from lettuce cultivar Regency 3.0 and to the lettuce plants, varieties, and their parts derived from the use of those methods. The invention further relates to hybrid lettuce seeds, plants, and plant parts produced by crossing cultivar Regency 3.0 with another lettuce cultivar.

BACKGROUND OF THE INVENTION

The present invention relates to a new iceberg lettuce (Latuca sativaL.) variety designated Regency 3.0. All publications cited in thisapplication are herein incorporated by reference.

There are numerous steps in the development of any novel, desirableplant germplasm. Plant breeding begins with the analysis and definitionof problems and weaknesses of the current germplasm, the establishmentof program goals, and the definition of specific breeding objectives.The next step is selection of germplasm that possess the traits to meetthe program goals. The goal is to combine in a single variety or hybridan improved combination of desirable traits from the parental germplasm.These important traits may include increased head size and weight,higher seed yield, improved color, resistance to diseases and insects,tolerance to drought and heat, and better agronomic quality.

Practically speaking, all cultivated forms of lettuce belong to thehighly polymorphic species Latuca sativa that is grown for its ediblehead and leaves. As a crop, lettuce is grown commercially whereverenvironmental conditions permit the production of an economically viableyield. Lettuce is the world's most popular salad. In the United States,the principal growing regions are California and Arizona; in 2013,California accounted for 71 percent of U.S. head lettuce production,followed by Arizona producing nearly 29 percent. According to the 2012USDA Census of Agriculture, lettuce was produced on 323,359 acres, whichwas up 3% since 2007. The value of U.S. lettuce production in 2013totaled nearly $1.5 billion, making lettuce the leading vegetable cropin terms of value. Fresh lettuce is available in the United Statesyear-round although the greatest supply is from May through October. Forplanting purposes, the lettuce season is typically divided into threecategories (i.e., early, mid, and late), with the coastal areas plantingfrom January to August, and the desert regions planting from August toDecember. Fresh lettuce is consumed nearly exclusively as fresh, rawproduct and occasionally as a cooked vegetable.

Latuca sativa is in the Cichoreae tribe of the Asteraceae (Compositae)family. Lettuce is related to chicory, sunflower, aster, dandelion,artichoke, and chrysanthemum. L. sativa is one of about 300 species inthe genus Lactuca. There are seven different morphological types oflettuce. The crisphead group includes the iceberg and batavian types.Iceberg lettuce has a large, firm head with a crisp texture and a whiteor creamy yellow interior. The batavian lettuce predates the icebergtype and has a smaller and less firm head. The butterhead group has asmall, soft head with an almost oily texture. The romaine, also known ascos lettuce, has elongated upright leaves forming a loose, loaf-shapedhead and the outer leaves are usually dark green. Leaf lettuce comes inmany varieties, none of which form a head, and include the green leafand green oak leaf varieties. Latin lettuce looks like a cross betweenromaine and butterhead. Stem lettuce has long, narrow leaves and thick,edible stems. Oilseed lettuce is a type grown for its large seeds thatare pressed to obtain oil. Latin lettuce, stem lettuce, and oilseedlettuce are seldom seen in the United States.

Lettuce in general is an important and valuable vegetable crop.Therefore, it is desirable to develop new varieties of lettuce havingnovel and exceptional traits, such as a combination of outstandingagronomic characteristics and resistance to diseases.

The foregoing examples of the related art and limitations relatedtherewith are intended to be illustrative and not exclusive. Otherlimitations of the related art will become apparent to those of skill inthe art upon a reading of the specification.

SUMMARY OF THE INVENTION

The following embodiments and aspects thereof are described inconjunction with systems, tools, and methods which are meant to beexemplary and illustrative, not limiting in scope. In variousembodiments, one or more of the above-described problems have beenreduced or eliminated, while other embodiments are directed to otherimprovements.

According to the invention, there is provided a novel lettuce cultivardesignated Regency 3.0. Also provided are lettuce plants having thephysiological and morphological characteristics of lettuce cultivarRegency 3.0. This invention thus relates to the seeds of lettucecultivar Regency 3.0, to the plants of lettuce cultivar Regency 3.0, andto methods for producing a lettuce plant produced by crossing thelettuce cultivar Regency 3.0 with itself or another lettuce plant, tomethods for producing a lettuce plant containing in its genetic materialone or more transgenes, and to the transgenic lettuce plants produced bythat method. This invention also relates to methods for producing otherlettuce cultivars derived from lettuce cultivar Regency 3.0 and to thelettuce cultivar derived by the use of those methods. This inventionfurther relates to hybrid lettuce seeds and plants produced by crossinglettuce cultivar Regency 3.0 with another lettuce variety.

In another aspect, the present invention provides regenerable cells foruse in tissue culture of lettuce cultivar Regency 3.0. The tissueculture will preferably be capable of regenerating plants havingessentially all of the physiological and morphological characteristicsof the foregoing lettuce plant, and of regenerating plants havingsubstantially the same genotype as the foregoing lettuce plant.Preferably, the regenerable cells in such tissue cultures will becallus, protoplasts, meristematic cells, cotyledons, hypocotyl, leaves,pollen, embryos, roots, root tips, anthers, pistils, shoots, stems,petiole flowers, stalks and seeds. Still further, the present inventionprovides lettuce plants regenerated from the tissue cultures of theinvention.

The invention also relates to methods for producing a lettuce plantcontaining in its genetic material one or more transgenes and to thetransgenic lettuce plant produced by those methods.

Another aspect of the current invention is a lettuce plant furthercomprising a single locus conversion. In one embodiment, the lettuceplant is defined as comprising the single locus conversion and otherwisecapable of expressing all of the morphological and physiologicalcharacteristics of the lettuce cultivar Regency 3.0. In particularembodiments of the invention, the single locus conversion may comprise atransgenic gene which has been introduced by genetic transformation intothe lettuce cultivar Regency 3.0 or a progenitor thereof. A transgenicor non-transgenic single locus conversion can also be introduced bybackcrossing, as is well known in the art. In still other embodiments ofthe invention, the single locus conversion may comprise a dominant orrecessive allele. The locus conversion may confer potentially any traitupon the single locus converted plant, including herbicide resistance,insect or pest resistance, resistance to bacterial, fungal, or viraldisease, modified fatty acid metabolism, modified carbohydratemetabolism, male fertility or sterility, improved nutritional quality,and industrial usage. The trait may be, for example, conferred by anaturally occurring gene introduced into the genome of the cultivar bybackcrossing, a natural or induced mutation, or a transgene introducedthrough genetic transformation techniques into the plant or a progenitorof any previous generation thereof. When introduced throughtransformation, a genetic locus may comprise one or more transgenesintegrated at a single chromosomal location.

The invention further relates to methods for genetically modifying alettuce plant of the lettuce cultivar Regency 3.0 and to the modifiedlettuce plant produced by those methods. The genetic modificationmethods may include, but are not limited to mutation, genome editing,RNA interference, gene silencing, backcross conversion, genetictransformation, single and multiple gene conversion, and/or direct genetransfer.

In still yet another aspect of the invention, the genetic complement ofthe lettuce cultivar Regency 3.0 is provided. The phrase “geneticcomplement” is used to refer to the aggregate of nucleotide sequences,the expression of which sequences defines the phenotype of, in thepresent case, a lettuce plant, or a cell or tissue of that plant. Agenetic complement thus represents the genetic makeup of a cell, tissueor plant, and a hybrid genetic complement represents the genetic makeupof a hybrid cell, tissue or plant. The invention thus provides lettuceplant cells that have a genetic complement in accordance with thelettuce plant cells disclosed herein, and plants, seeds and plantscontaining such cells. Plant genetic complements may be assessed bygenetic marker profiles, and by the expression of phenotypic traits thatare characteristic of the expression of the genetic complement, e.g.,isozyme typing profiles.

In still yet another aspect, the invention provides a method ofdetermining the genotype of a plant of lettuce cultivar Regency 3.0comprising detecting in the genome of the plant at least a firstpolymorphism. The method may, in certain embodiments, comprise detectinga plurality of polymorphisms in the genome of the plant. The method mayfurther comprise storing the results of the step of detecting theplurality of polymorphisms on a computer readable medium. The inventionfurther provides a computer readable medium produced by such a method.

This invention further relates to the F₁ hybrid lettuce plants and plantparts grown from the hybrid seed produced by crossing lettuce cultivarRegency 3.0 to a second lettuce plant. Still further included in theinvention are the seeds of an F₁ hybrid plant produced with the lettucecultivar Regency 3.0 as one parent, the second generation (F₂) hybridlettuce plant grown from the seed of the F₁ hybrid plant, and the seedsof the F₂ hybrid plant. Thus, any such methods using the lettucecultivar Regency 3.0 are part of this invention: selfing, backcrosses,hybrid production, crosses to populations, and the like. All plantsproduced using lettuce cultivar Regency 3.0 as at least one parent arewithin the scope of this invention. Advantageously, the lettuce cultivarcould be used in crosses with other, different, lettuce plants toproduce first generation (F₁) lettuce hybrid seeds and plants withsuperior characteristics.

The invention further provides methods for developing lettuce plants ina lettuce plant breeding program using plant breeding techniquesincluding but not limited to recurrent selection, backcrossing, pedigreebreeding, restriction fragment length polymorphism enhanced selection,genetic marker enhanced selection, and transformation. Seeds, lettuceplants, and parts thereof, produced by such breeding methods are alsopart of the invention.

This invention also relates to lettuce plants or breeding cultivars andplant parts derived from lettuce cultivar Regency 3.0. Still yet anotheraspect of the invention is a method of producing a lettuce plant derivedfrom the lettuce cultivar Regency 3.0, the method comprising the stepsof: (a) preparing a progeny plant derived from lettuce cultivar Regency3.0 by crossing a plant of the lettuce cultivar Regency 3.0 with asecond lettuce plant; and (b) crossing the progeny plant with itself ora second plant to produce a seed of a progeny plant of a subsequentgeneration which is derived from a plant of the lettuce cultivar Regency3.0. In further embodiments of the invention, the method mayadditionally comprise: (c) growing a progeny plant of a subsequentgeneration from said seed of a progeny plant of a subsequent generationand crossing the progeny plant of a subsequent generation with itself ora second plant; and repeating the steps for an additional 2-10generations to produce a lettuce plant derived from the lettuce cultivarRegency 3.0. The plant derived from lettuce cultivar Regency 3.0 may bean inbred line, and the aforementioned repeated crossing steps may bedefined as comprising sufficient inbreeding to produce the inbred line.In the method, it may be desirable to select particular plants resultingfrom step (c) for continued crossing according to steps (b) and (c). Byselecting plants having one or more desirable traits, a plant derivedfrom lettuce cultivar Regency 3.0 is obtained which possesses some ofthe desirable traits of the line as well as potentially other selectedtraits. Also provided by the invention is a plant produced by this andthe other methods of the invention.

In another embodiment of the invention, the method of producing alettuce plant derived from the lettuce cultivar Regency 3.0 furthercomprises: (a) crossing the lettuce cultivar Regency 3.0-derived lettuceplant with itself or another lettuce plant to yield additional lettucecultivar Regency 3.0-derived progeny lettuce seed; (b) growing theprogeny lettuce seed of step (a) under plant growth conditions to yieldadditional lettuce cultivar Regency 3.0-derived lettuce plants; and (c)repeating the crossing and growing steps of (a) and (b) to generatefurther lettuce cultivar Regency 3.0-derived lettuce plants. In specificembodiments, steps (a) and (b) may be repeated at least 1, 2, 3, 4, or 5or more times as desired. The invention still further provides a lettuceplant produced by this and the foregoing methods.

The invention also provides methods of multiplication or propagation oflettuce plants of the invention, which can be accomplished using anymethod known in the art, for example, via vegetative propagation and/orseed. Still further, as another aspect, the invention provides a methodof vegetatively propagating a plant of lettuce cultivar Regency 3.0. Ina non-limiting example, the method comprises: (a) collecting a plantpart capable of being propagated from a plant of lettuce cultivarRegency 3.0; (b) producing at least a first rooted plant from said plantpart. The invention also encompasses the plantlets and plants producedby these methods.

The invention further relates to a method of producing a commodity plantproduct from lettuce cultivar Regency 3.0, such as fresh lettuce leaf,fresh lettuce head, cut, sliced, ground, pureed, dried, canned, jarred,washed, packaged, frozen and/or heated leaves, and to the commodityplant product produced by the method.

In addition to the exemplary aspects and embodiments described above,further aspects and embodiments will become apparent by reference bystudy of the following descriptions.

DETAILED DESCRIPTION OF THE INVENTION

In the description and tables which follow, a number of terms are used.In order to provide a clear and consistent understanding of thespecification and claims, including the scope to be given such terms,the following definitions are provided:

Abiotic stress. As used herein, abiotic stress relates to all non-livingchemical and physical factors in the environment. Examples of abioticstress include, but are not limited to, drought, flooding, salinity,temperature, and climate change.

Allele. The allele is any of one or more alternative forms of a gene,all of which relate to one trait or characteristic. In a diploid cell ororganism, the two alleles of a given gene occupy corresponding loci on apair of homologous chromosomes.

Alter. The utilization of up-regulation, down-regulation, or genesilencing.

Backcrossing. A process in which a breeder crosses progeny back to oneof the parental genotypes one or more times. Commonly used to introduceone or more locus conversions from one genetic background into another(backcross conversion).

Bolting. The premature development of a flowering stalk, and subsequentseed, before a plant produces a food crop. Bolting is typically causedby late planting when temperatures are low enough to cause vernalizationof the plants.

Bremia lactucae. An Oomycete that causes downy mildew in lettuce incooler growing regions.

Cell. Cell as used herein includes a plant cell, whether isolated, intissue culture or incorporated in a plant or plant part. The cell can bea cell, such as a somatic cell, of the variety having the same set ofchromosomes as the cells of the deposited seed, or, if the cell containsa locus conversion or transgene, otherwise having the same oressentially the same set of chromosomes as the cells of the depositedseed.

Core diameter. The diameter of the lettuce stem at the base of the cuthead.

Core length. Length of the internal lettuce stem measured from the baseof the cut and trimmed head to the tip of the stem.

Corky root. A disease caused by the bacterium Rhizomonas suberifaciens,which causes the entire taproot to become brown, severely cracked, andnon-functional.

Cotyledon. One of the first leaves of the embryo of a seed plant;typically one or more in monocotyledons, two in dicotyledons, and two ormore in gymnosperms.

Essentially all of the physiological and morphological characteristics.A plant having essentially all of the physiological and morphologicalcharacteristics of a designated plant has all of the characteristics ofthe plant that are otherwise present when compared in the sameenvironment, other than an occasional variant trait that might ariseduring backcrossing or direct introduction of a transgene.

F_(#). The “F” symbol denotes the filial generation, and the # is thegeneration number, such as F₁, F₂, F₃, etc.

F₁ Hybrid. The first generation progeny of the cross of two nonisogenicplants.

First water date. The date the seed first receives adequate moisture togerminate. This can and often does equal the planting date.

Frame diameter. The frame diameter is a measurement of the lettuce plantdiameter at its widest point, measured from the outer most wrapper leaftip to the outer most wrapper leaf tip.

Fusarium oxysporum. Fusarium wilt of lettuce is caused by the soil-bornefungus Fusarium oxysporum f. sp. lactucae. There are three reportedraces of Fusarium oxysporum f. sp. lactucae. All three races are presentin Japan, whereas only race 1 is known to occur in the United States(Arizona and California). Infection results in yellowing and necrosis ofleaves, as well as stunted, wilted plants and often plant death.

Gene. As used herein, “gene” refers to a segment of nucleic acid. A genecan be introduced into a genome of a species, whether from a differentspecies or from the same species, using transformation or variousbreeding methods.

Gene silencing. The interruption or suppression of the expression of agene at the level of transcription or translation.

Genetically modified. Describes an organism that has received geneticmaterial from another organism, or had its genetic material modified,resulting in a change in one or more of its phenotypic characteristics.Methods used to modify, introduce or delete the genetic material mayinclude mutation breeding, genome editing, RNA interference, genesilencing, backcross conversion, genetic transformation, single andmultiple gene conversion, and/or direct gene transfer.

Genome editing. A type of genetic engineering in which DNA is inserted,replaced, modified or removed from a genome using artificiallyengineered nucleases or other targeted changes using homologousrecombination. Examples include but are not limited to use of zincfinger nucleases (ZFNs), TAL effector nucleases (TALENs), meganucleases,CRISPR/Cas9, and other CRISPR related technologies. (Ma et. al.,Molecular Plant, 9:961-974 (2016); Belhaj et. al., Current Opinion inBiotechnology, 32:76-84 (2015)).

Genotype. Refers to the genetic constitution of a cell or organism.

Green leaf lettuce. A type of lettuce characterized by having curled orincised leaves forming a loose green rosette that does not develop intoa compact head.

Haploid. A cell or organism having one set of the two sets ofchromosomes in a diploid.

Head diameter. Diameter of the cut and trimmed head, sliced vertically,and measured at the widest point perpendicular to the stem.

Head height. Height of the cut and trimmed head, sliced vertically, andmeasured from the base of the cut stem to the cap leaf.

Head weight. Weight of saleable lettuce head, cut and trimmed to marketspecifications.

Iceberg lettuce. A type of lettuce characterized by having a large, firmhead with a crisp texture and a white or creamy yellow interior.

Lettuce big vein virus (LBV). Big vein is a disease of lettuce caused bylettuce mirafiori big vein virus which is transmitted by the fungusOlpidium virulentus, with vein clearing and leaf shrinkage resulting inplants of poor quality and reduced marketable value.

Lettuce mosaic virus. A disease that can cause a stunted, deformed, ormottled pattern in young lettuce and yellow, twisted, and deformedleaves in older lettuce.

Lettuce necrotic stunt virus (LNSV). A disease of lettuce that can causeseverely stunted plants having yellowed outer leaves and brown, necroticspotting. LNSV is a soil-borne virus from the Tombusvirus family with noknown vector.

Linkage. Refers to a phenomenon wherein alleles on the same chromosometend to segregate together more often than expected by chance if theirtransmission was independent.

Linkage disequilibrium. Refers to a phenomenon wherein alleles tend toremain together in linkage groups when segregating from parents tooffspring, with a greater frequency than expected from their individualfrequencies.

Locus. A defined segment of DNA.

Locus conversion (also called a ‘trait conversion’ or ‘geneconversion’). A locus conversion refers to a plant or plants within avariety or line that have been modified in a manner that retains theoverall genetics of the variety and further comprises one or more lociwith a specific desired trait, such as but not limited to malesterility, insect or pest control, disease control or herbicidetolerance. Examples of single locus conversions include mutant genes,transgenes and native traits finely mapped to a single locus. One ormore locus conversion traits may be introduced into a single cultivar.

Market stage. Market stage is the stage when a lettuce plant is readyfor commercial lettuce harvest. In the case of an iceberg variety, thehead is solid, and has reached an adequate size and weight.

Maturity date. Maturity refers to the stage when the plants are of fullsize or optimum weight, in marketable form or shape to be of commercialor economic value.

Nasonovia ribisnigri. A lettuce aphid that colonizes the innermostleaves of the lettuce plant, contaminating areas that cannot be treatedeasily with insecticides.

Pedigree. Refers to the lineage or genealogical descent of a plant.

Pedigree distance. Relationship among generations based on theirancestral links as evidenced in pedigrees. May be measured by thedistance of the pedigree from a given starting point in the ancestry.

Plant. “Plant” includes plant cells, plant protoplasts, plant tissue,plant cells of tissue culture from which lettuce plants can beregenerated, plant calli, plant clumps and plant cells that are intactin plants, or parts of plants such as pollen, flowers, seeds, leaves,stems and the like.

Plant part. Includes any part, organ, tissue or cell of a plantincluding without limitation an embryo, meristem, leaf, pollen,cotyledon, hypocotyl, root, root tip, anther, flower, flower bud,pistil, ovule, seed, shoot, stem, stalk, petiole, pith, capsule, ascion, a rootstock and/or a fruit including callus and protoplastsderived from any of the foregoing.

Quantitative Trait Loci. Quantitative Trait Loci (QTL) refers to geneticloci that control to some degree, numerically representable traits thatare usually continuously distributed.

Ratio of head height/diameter. Head height divided by the head diameteris an indication of the head shape; <1 is flattened, 1=round, and >1 ispointed.

Regeneration. Regeneration refers to the development of a plant fromtissue culture.

RHS. RHS refers to the Royal Horticultural Society of England whichpublishes an official botanical color chart quantitatively identifyingcolors according to a defined numbering system. The chart may bepurchased from Royal Horticulture Society Enterprise Ltd., RHS Garden;Wisley, Woking; Surrey GU236QB, UK.

Rogueing. Rogueing is the process in seed production where undesiredplants are removed from a variety. The plants are removed since theydiffer physically from the general desired expressed characteristics ofthe variety. The differences can be related to size, color, maturity,leaf texture, leaf margins, growth habit, or any other characteristicthat distinguishes the plant.

Romaine lettuce. A lettuce variety having elongated upright leavesforming a loose, loaf-shaped head and the outer leaves are usually darkgreen.

Sclerotinia sclerotiorum. A plant pathogenic fungus that can cause adisease called white mold. Also known as cottony rot, watery soft rot,stem rot, drop, crown rot and blossom blight.

Single locus converted (conversion) plant. Plants which are developed bya plant breeding technique called backcrossing or via geneticengineering wherein essentially all of the morphological andphysiological characteristics of a variety are recovered in addition tothe desired trait or characteristics conferred by the single locustransferred into the variety via the backcrossing technique or viagenetic engineering. A single locus may comprise one gene, or in thecase of transgenic plants, one or more transgenes integrated into thehost genome at a single site (locus).

Tipburn. Means a browning of the edges or tips of lettuce leaves thathas an unknown cause, possibly a calcium deficiency.

Tomato Bushy Stunt. A disease which causes stunting of growth, leafmottling, and deformed or absent fruit.

Transgene. A nucleic acid of interest that can be introduced into thegenome of a plant by genetic engineering techniques (e.g.,transformation) or breeding.

The following detailed description is of the currently contemplatedmodes of carrying out the invention. The description is not to be takenin a limiting sense, but is made merely for the purpose of illustratingthe general principles of the invention, since the scope of theinvention is best defined by the appended claims.

Lettuce cultivar Regency 3.0 is a novel iceberg lettuce variety that hasa large head size and resistance to downy mildew (CA I-IX and novels),corky root (CA-I) and lettuce necrotic stunt virus (LNSV). The iceberglettuce variety exemplified in the present invention, Regency 3.0, isdifferent from known varieties of iceberg lettuce in having anunexpected and unique combination of traits. Lettuce cultivar Regency3.0 is adapted to the summer and autumn growing region in SalinasValley, Santa Maria, Calif. Additionally, lettuce cultivar Regency 3.0is highly resistant to tipburn and is moderately resistant toSclerotinia sclerotiorum.

Lettuce cultivar Regency 3.0 has shown uniformity and stability for thetraits, within the limits of environmental influence for the traits. Ithas been self-pollinated a sufficient number of generations with carefulattention to uniformity of plant type. The line has been increased withcontinued observation for uniformity. No variant traits have beenobserved or are expected in cultivar Regency 3.0.

Lettuce cultivar Regency 3.0 has the following morphological andphysiological characteristics described (based on data primarilycollected in California):

TABLE 1 VARIETY DESCRIPTION INFORMATION Plant: Type: Iceberg lettuceDays to maturity: 75 Seed: Color: Black Light dormancy: Absent Heatdormancy: Present Cotyledon (to fourth leaf stage): Shape: SpatulateShape of fourth leaf: Elongated Length/width index of 4^(th) leaf (L/W ×10): 15 Apex: Moderately dentate Base: Coarsely dentate Undulation:Medium Green color: Dark green Anthocyanin distribution: Absent Rolling:Present Cupping: Uncupped Reflexing: Apical margin Mature Leaves:Margin: Incision depth: Moderate Indentation: Crenate Undulation of theapical margin: Moderate Green color (at harvest maturity): Medium greenAnthocyanin distribution: Absent Blistering: Moderate Glossiness:Moderate Thickness: Thick Trichomes: Absent Plant (at market stage):Spread of frame leaves: 40.0 cm Head diameter: 15.0 cm Head shape:Spherical Head size class: Large Head weight: 750.0 g Head firmness:Very firm Butt: Shape: Rounded Midrib: Flattened Core: Diameter at baseof head: 36.0 mm Core height from base of head to apex: 36.0 mm Bolting:Number of days from first water date to seed stalk emergence: 54 Time ofbeginning of bolting: Mid-summer Class: Medium Height of mature seedstalk: 99.0 cm Spread of bolter plant: 42.0 cm Bolter leaves: StraightBolter leaf margin: Dentate Bolter leaf color: Medium green Bolterhabit: Terminal inflorescence: Present Lateral shoots: Absent Basal sideshoots: Absent Primary Regions of Adaptation: Spring area: Not adaptedSummer area: Salinas Valley, Santa Maria, California Autumn area:Salinas Valley, Santa Maria, California Winter area: Not adaptedDisease/Pest Resistance: Lettuce mosaic virus: Not tested Downy mildew(Bremia lactucae): Resistant (CAI-IX and novels) Lettuce necrotic stuntvirus (LNSV): Resistant Corky root (Rhizomonas suberifaciens): Resistant(CA-I) Sclerotinia sclerotiorum: Moderately resistant Lettuce aphid(Nasonovia ribisnigri): Susceptible to Nr0 and Nr1 PhysiologicalResponses: Tipburn: Highly resistant

Further Embodiments of the Invention

Lettuce in general, and iceberg lettuce in particular, is an importantand valuable vegetable crop. Thus, a continuing goal of lettuce plantbreeders is to develop stable, high yielding lettuce cultivars that areagronomically sound. To accomplish this goal, the lettuce breeder mustselect and develop lettuce plants with traits that result in superiorcultivars.

Plant breeding techniques known in the art and used in a lettuce plantbreeding program include, but are not limited to, pedigree breeding,recurrent selection, mass selection, single or multiple-seed descent,bulk selection, backcrossing, open pollination breeding, restrictionfragment length polymorphism enhanced selection, genetic marker enhancedselection, making double haploids, and transformation. Oftencombinations of these techniques are used. The development of lettucevarieties in a plant breeding program requires, in general, thedevelopment and evaluation of homozygous varieties. There are manyanalytical methods available to evaluate a new variety. The oldest andmost traditional method of analysis is the observation of phenotypictraits, but genotypic analysis may also be used.

Using Lettuce Cultivar Regency 3.0 to Develop Other Lettuce Varieties

This invention is directed to methods for producing a lettuce plant bycrossing a first parent lettuce plant with a second parent lettuce plantwherein either the first or second parent lettuce plant is varietyRegency 3.0. Also provided are methods for producing a lettuce planthaving substantially all of the morphological and physiologicalcharacteristics of cultivar Regency 3.0, by crossing a first parentlettuce plant with a second parent lettuce plant wherein the firstand/or the second parent lettuce plant is a plant having substantiallyall of the morphological and physiological characteristics of cultivarRegency 3.0 set forth in Table 1, as determined at the 5% significancelevel when grown in the same environmental conditions. The other parentmay be any lettuce plant, such as a lettuce plant that is part of asynthetic or natural population. Any such methods using lettuce cultivarRegency 3.0 include but are not limited to selfing, sibbing,backcrossing, mass selection, pedigree breeding, bulk selection, hybridproduction, crossing to populations, and the like. These methods arewell known in the art and some of the more commonly used breedingmethods are described below. Descriptions of breeding methods can befound in one of several reference books (e.g., Allard, Principles ofPlant Breeding, 1960; Simmonds, Principles of Crop Improvement, 1979;Fehr, “Breeding Methods for Cultivar Development”, Chapter 7, LettuceImprovement, Production and Uses, 2.sup.nd ed., Wilcox editor, 1987).

Another method involves producing a population of lettuce cultivarRegency 3.0 progeny lettuce plants, comprising crossing variety Regency3.0 with another lettuce plant, thereby producing a population oflettuce plants which, on average, derive 50% of their alleles fromlettuce cultivar Regency 3.0. A plant of this population may be selectedand repeatedly selfed or sibbed with a lettuce cultivar resulting fromthese successive filial generations. One embodiment of this invention isthe lettuce cultivar produced by this method and that has obtained atleast 50% of its alleles from lettuce cultivar Regency 3.0.

One of ordinary skill in the art of plant breeding would know how toevaluate the traits of two plant varieties to determine if there is nosignificant difference between the two traits expressed by thosevarieties. For example, see, Fehr and Walt, Principles of CultivarDevelopment, pp. 261-286 (1987). Thus the invention includes lettucecultivar Regency 3.0 progeny lettuce plants comprising a combination ofat least two of variety Regency 3.0 traits selected from the groupconsisting of those listed in Table 1, or the variety Regency 3.0combination of traits listed in the Detailed Description of theInvention, so that said progeny lettuce plant is not significantlydifferent for said traits than lettuce cultivar Regency 3.0 asdetermined at the 5% significance level when grown in the sameenvironmental conditions. Using techniques described herein, molecularmarkers may be used to identify said progeny plant as a lettuce cultivarRegency 3.0 progeny plant. Mean trait values may be used to determinewhether trait differences are significant, and preferably the traits aremeasured on plants grown under the same environmental conditions. Oncesuch a variety is developed, its value is substantial since it isimportant to advance the germplasm base as a whole in order to maintainor improve traits such as yield, disease resistance, pest resistance,and plant performance in extreme environmental conditions.

The goal of lettuce plant breeding is to develop new, unique, andsuperior lettuce cultivars. The breeder initially selects and crossestwo or more parental lines, followed by repeated selfing and selection,producing many new genetic combinations. The breeder can theoreticallygenerate billions of different genetic combinations via crossing,selfing, and mutations. The breeder has no direct control at thecellular level and the cultivars that are developed are unpredictable.This unpredictability is because the breeder's selection occurs inunique environments, with no control at the DNA level (usingconventional breeding procedures), and with millions of differentpossible genetic combinations being generated. A breeder of ordinaryskill in the art cannot predict the final resulting lines he develops,except possibly in a very gross and general fashion. The same breedercannot produce the same line twice by using the exact same originalparents and the same selection techniques. Therefore, two breeders willnever develop the same line, or even very similar lines, having the samelettuce traits.

Progeny of lettuce cultivar Regency 3.0 may also be characterizedthrough their filial relationship with lettuce cultivar Regency 3.0, asfor example, being within a certain number of breeding crosses oflettuce cultivar Regency 3.0. A breeding cross is a cross made tointroduce new genetics into the progeny, and is distinguished from across, such as a self or a sib cross, made to select among existinggenetic alleles. The lower the number of breeding crosses in thepedigree, the closer the relationship between lettuce cultivar Regency3.0 and its progeny. For example, progeny produced by the methodsdescribed herein may be within 1, 2, 3, 4, or 5 breeding crosses oflettuce cultivar Regency 3.0.

Pedigree breeding starts with the crossing of two genotypes, such aslettuce cultivar Regency 3.0 or a lettuce variety having all of themorphological and physiological characteristics of Regency 3.0, andanother lettuce variety having one or more desirable characteristicsthat is lacking or which complements lettuce cultivar Regency 3.0. Ifthe two original parents do not provide all the desired characteristics,other sources can be included in the breeding population. In thepedigree method, superior plants are selfed and selected in successivefilial generations. In the succeeding filial generations, theheterozygous condition gives way to the homozygous allele condition as aresult of inbreeding. Typically in the pedigree method of breeding, fiveor more successive filial generations of selfing and selection ispracticed: F₁ to F₂; F₂ to F₃; F₃ to F₄; F₄ to F₅; etc. In someexamples, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more generations of selfingand selection are practiced. After a sufficient amount of inbreeding,successive filial generations will serve to increase seed of thedeveloped variety. Preferably, the developed variety compriseshomozygous alleles at about 95% or more of its loci.

In addition to being used to create backcross conversion populations,backcrossing can also be used in combination with pedigree breeding. Asdiscussed previously, backcrossing can be used to transfer one or morespecifically desirable traits from one variety (the donor parent) to adeveloped variety (the recurrent parent), which has good overallagronomic characteristics yet may lack one or more other desirabletraits. However, the same procedure can be used to move the progenytoward the genotype of the recurrent parent but at the same time retainmany components of the non-recurrent parent by stopping the backcrossingat an early stage and proceeding with selfing and selection. Forexample, a lettuce variety may be crossed with another variety toproduce a first generation progeny plant. The first generation progenyplant may then be backcrossed to one of its parent varieties to create aBC₁F₁. Progeny are selfed and selected so that the newly developedvariety has many of the attributes of the recurrent parent and yetseveral of the desired attributes of the donor parent. This approachleverages the value and strengths of both parents for use in new lettucevarieties.

Therefore, in some examples a method of making a backcross conversion oflettuce cultivar Regency 3.0, comprising the steps of crossing a plantof lettuce cultivar Regency 3.0 or a lettuce variety having all of themorphological and physiological characteristics of Regency 3.0 with adonor plant possessing a desired trait to introduce the desired trait,selecting an F₁ progeny plant containing the desired trait, andbackcrossing the selected F₁ progeny plant to a plant of lettucecultivar Regency 3.0 are provided. This method may further comprise thestep of obtaining a molecular marker profile of lettuce cultivar Regency3.0 and using the molecular marker profile to select for a progeny plantwith the desired trait and the molecular marker profile of Regency 3.0.The molecular marker profile can comprise information from one or moremarkers. In one example the desired trait is a mutant gene or transgenepresent in the donor parent. In another example, the desired trait is anative trait in the donor parent.

Mass and recurrent selections can be used to improve populations ofeither self- or cross-pollinating crops. A genetically variablepopulation of heterozygous individuals is either identified or createdby intercrossing several different parents. The best plants are selectedbased on individual superiority, outstanding progeny, or excellentcombining ability. The selected plants are intercrossed to produce a newpopulation in which further cycles of selection are continued.

The single-seed descent procedure in the strict sense refers to plantinga segregating population, harvesting a sample of one seed per plant, andusing the one-seed sample to plant the next generation. When thepopulation has been advanced from the F₂ to the desired level ofinbreeding, the plants from which lines are derived will each trace todifferent F₂ individuals. The number of plants in a population declineswith each generation due to failure of some seeds to germinate or someplants to produce at least one seed. As a result, not all of the F₂plants originally sampled in the population will be represented by aprogeny when generation advance is completed.

Mutation breeding is another method of introducing new traits intolettuce varieties. Mutations that occur spontaneously or areartificially induced can be useful sources of variability for a plantbreeder. The goal of artificial mutagenesis is to increase the rate ofmutation for a desired characteristic. Mutation rates can be increasedby many different means including temperature, long-term seed storage,tissue culture conditions, radiation; such as X-rays, Gamma rays (e.g.,cobalt 60 or cesium 137), neutrons, (product of nuclear fission byuranium 235 in an atomic reactor), Beta radiation (emitted fromradioisotopes such as phosphorus 32 or carbon 14), or ultravioletradiation (preferably from 2500 to 2900 nm), or chemical mutagens (suchas base analogues (5-bromo-uracil)), related compounds (8-ethoxycaffeine), antibiotics (streptonigrin), alkylating agents (sulfurmustards, nitrogen mustards, epoxides, ethylenamines, sulfates,sulfonates, sulfones, lactones), azide, hydroxylamine, nitrous acid, oracridines. Once a desired trait is observed through mutagenesis thetrait may then be incorporated into existing germplasm by traditionalbreeding techniques. Details of mutation breeding can be found in Fehr,“Principles of Cultivar Development,” Macmillan Publishing Company(1993). In addition, mutations created in other lettuce plants may beused to produce a backcross conversion of lettuce cultivar Regency 3.0that comprises such mutation.

Selection of lettuce plants for breeding is not necessarily dependent onthe phenotype of a plant and instead can be based on geneticinvestigations. For example, one may utilize a suitable genetic markerwhich is closely associated with a trait of interest. One of thesemarkers may therefore be used to identify the presence or absence of atrait in the offspring of a particular cross, and hence may be used inselection of progeny for continued breeding. This technique may commonlybe referred to as marker assisted selection. Any other type of geneticmarker or other assay which is able to identify the relative presence orabsence of a trait of interest in a plant may also be useful forbreeding purposes. Procedures for marker assisted selection applicableto the breeding of lettuces are well known in the art. Such methods willbe of particular utility in the case of recessive traits and variablephenotypes, or where conventional assays may be more expensive, timeconsuming or otherwise disadvantageous. Types of genetic markers whichcould be used in accordance with the invention include, but are notnecessarily limited to, Isozyme Electrophoresis, Restriction FragmentLength Polymorphisms (RFLPs), Simple Sequence Length Polymorphisms(SSLPs) (Williams et al., Nucleic Acids Res., 18:6531-6535, 1990),Randomly Amplified Polymorphic DNAs (RAPDs), DNA AmplificationFingerprinting (DAF), Sequence Characterized Amplified Regions (SCARs),Arbitrary Primed Polymerase Chain Reaction (AP-PCR), Amplified FragmentLength Polymorphisms (AFLPs) (EP 534 858, specifically incorporatedherein by reference in its entirety), Simple Sequence Repeats (SSRs),and Single Nucleotide Polymorphisms (SNPs) (Wang et al., Science,280:1077-1082, 1998).

The production of double haploids can also be used for the developmentof homozygous varieties in a breeding program. Double haploids areproduced by the doubling of a set of chromosomes from a heterozygousplant to produce a completely homozygous individual. For example, see,Wan, et al., “Efficient Production of Doubled Haploid Plants ThroughColchicine Treatment of Anther-Derived Maize Callus,” Theoretical andApplied Genetics, 77:889-892 (1989) and U.S. Pat. No. 7,135,615. Thiscan be advantageous because the process omits the generations of selfingneeded to obtain a homozygous plant from a heterozygous source.

Descriptions of other breeding methods that are commonly used fordifferent traits and crops can be found in one of several referencebooks (e.g., Allard, “Principles of plant breeding,” John Wiley & Sons,NY, University of California, Davis, Calif., 50-98, 1960; Simmonds,“Principles of crop improvement,” Longman, Inc., NY, 369-399, 1979;Sneep and Hendriksen, “Plant breeding perspectives,” Wageningen (ed),Center for Agricultural Publishing and Documentation, 1979; Fehr, In:Soybeans: Improvement, Production and Uses,” 2d Ed., Manograph 16:249,1987; Fehr, “Principles of cultivar development,” Theory and Technique(Vol 1) and Crop Species Soybean (Vol 2), Iowa State Univ., MacmillianPub. Co., NY, 360-376, 1987; Poehlman and Sleper, “Breeding Field Crops”Iowa State University Press, Ames, 1995; Sprague and Dudley, eds., Cornand Improvement, 5th ed., 2006).

Genotypic Profile of Regency 3.0 and Progeny

In addition to phenotypic observations, a plant can also be identifiedby its genotype. The genotype of a plant can be characterized through agenetic marker profile which can identify plants of the same variety ora related variety, or which can be used to determine or validate apedigree. Genetic marker profiles can be obtained by techniques such asrestriction fragment length polymorphisms (RFLPs), randomly amplifiedpolymorphic DNAs (RAPDs), arbitrarily primed polymerase chain reaction(AP-PCR), DNA amplification fingerprinting (DAF), sequence characterizedamplified regions (SCARs), amplified fragment length polymorphisms(AFLPs), simple sequence repeats (SSRs) also referred to asmicrosatellites, single nucleotide polymorphisms (SNPs), or genome-wideevaluations such as genotyping-by-sequencing (GBS). For example, seeCregan et al. (1999) “An Integrated Genetic Linkage Map of the SoybeanGenome” Crop Science 39:1464-1490, and Berry et al. (2003) “AssessingProbability of Ancestry Using Simple Sequence Repeat Profiles:Applications to Maize Inbred Lines and Soybean Varieties” Genetics165:331-342, each of which are incorporated by reference herein in theirentirety. Favorable genotypes and or marker profiles, optionallyassociated with a trait of interest, may be identified by one or moremethodologies.

In some examples one or more markers are used, including but not limitedto AFLPs, RFLPs, ASH, SSRs, SNPs, indels, padlock probes, molecularinversion probes, microarrays, sequencing, and the like. In somemethods, a target nucleic acid is amplified prior to hybridization witha probe. In other cases, the target nucleic acid is not amplified priorto hybridization, such as methods using molecular inversion probes (see,for example Hardenbol et al. (2003) Nat Biotech 21:673-678). In someexamples, the genotype related to a specific trait is monitored, whilein other examples, a genome-wide evaluation including but not limited toone or more of marker panels, library screens, association studies,microarrays, gene chips, expression studies, or sequencing such aswhole-genome resequencing and genotyping-by-sequencing (GBS) may beused. In some examples, no target-specific probe is needed, for exampleby using sequencing technologies, including but not limited tonext-generation sequencing methods (see, for example, Metzker (2010) NatRev Genet 11:31-46; and, Egan et al. (2012) Am J Bot 99:175-185) such assequencing by synthesis (e.g., Roche 454 pyrosequencing, Illumina GenomeAnalyzer, and Ion Torrent PGM or Proton systems), sequencing by ligation(e.g., SOLiD from Applied Biosystems, and Polnator system from AzcoBiotech), and single molecule sequencing (SMS or third-generationsequencing) which eliminate template amplification (e.g., Helicossystem, and PacBio RS system from Pacific BioSciences). Furthertechnologies include optical sequencing systems (e.g., Starlight fromLife Technologies), and nanopore sequencing (e.g., GridION from OxfordNanopore Technologies). Each of these may be coupled with one or moreenrichment strategies for organellar or nuclear genomes in order toreduce the complexity of the genome under investigation via PCR,hybridization, restriction enzyme (see, e.g., Elshire et al. (2011) PLoSONE 6:e19379), and expression methods. In some examples, no referencegenome sequence is needed in order to complete the analysis.

The invention further provides a method of determining the genotype of aplant of lettuce cultivar Regency 3.0, or a first generation progenythereof, which may comprise obtaining a sample of nucleic acids fromsaid plant and detecting in said nucleic acids a plurality ofpolymorphisms. This method may additionally comprise the step of storingthe results of detecting the plurality of polymorphisms on a computerreadable medium. The plurality of polymorphisms are indicative of and/orgive rise to the expression of the morphological and physiologicalcharacteristics of lettuce cultivar Regency 3.0.

With any of the genotyping techniques mentioned herein, polymorphismsmay be detected when the genotype and/or sequence of the plant ofinterest is compared to the genotype and/or sequence of one or morereference plants. The polymorphism revealed by these techniques may beused to establish links between genotype and phenotype. Thepolymorphisms may thus be used to predict or identify certain phenotypiccharacteristics, individuals, or even species. The polymorphisms aregenerally called markers. It is common practice for the skilled artisanto apply molecular DNA techniques for generating polymorphisms andcreating markers. The polymorphisms of this invention may be provided ina variety of mediums to facilitate use, e.g. a database or computerreadable medium, which may also contain descriptive annotations in aform that allows a skilled artisan to examine or query the polymorphismsand obtain useful information.

In some examples, a plant, a plant part, or a seed of lettuce cultivarRegency 3.0 may be characterized by producing a molecular profile. Amolecular profile may include, but is not limited to, one or moregenotypic and/or phenotypic profile(s). A genotypic profile may include,but is not limited to, a marker profile, such as a genetic map, alinkage map, a trait maker profile, a SNP profile, an SSR profile, agenome-wide marker profile, a haplotype, and the like. A molecularprofile may also be a nucleic acid sequence profile, and/or a physicalmap. A phenotypic profile may include, but is not limited to, a proteinexpression profile, a metabolic profile, an mRNA expression profile, andthe like.

SSR technology is currently the most efficient and practical markertechnology; more marker loci can be routinely used and more alleles permarker locus can be found using SSRs in comparison to RFLPs. Forexample, Diwan and Cregan described a highly polymorphic microsatellitelocus in soybean with as many as 26 alleles. Diwan, N. and Cregan, P.B., Theor. Appl. Genet., 95:22-225 (1997). SNPs may also be used toidentify the unique genetic composition of the invention and progenyvarieties retaining that unique genetic composition. Various molecularmarker techniques may be used in combination to enhance overallresolution.

Molecular markers, which include markers identified through the use oftechniques such as Isozyme Electrophoresis, RFLPs, RAPDs, AP-PCR, DAF,SCARs, AFLPs, SSRs, and SNPs, may be used in plant breeding. One use ofmolecular markers is Quantitative Trait Loci (QTL) mapping. QTL mappingis the use of markers which are known to be closely linked to allelesthat have measurable effects on a quantitative trait. Selection in thebreeding process is based upon the accumulation of markers linked to thepositive effecting alleles and/or the elimination of the markers linkedto the negative effecting alleles from the plant's genome.

Molecular markers can also be used during the breeding process for theselection of qualitative traits. For example, markers closely linked toalleles or markers containing sequences within the actual alleles ofinterest can be used to select plants that contain the alleles ofinterest during a backcrossing breeding program. The markers can also beused to select toward the genome of the recurrent parent and against themarkers of the donor parent. This procedure attempts to minimize theamount of genome from the donor parent that remains in the selectedplants. It can also be used to reduce the number of crosses back to therecurrent parent needed in a backcrossing program. The use of molecularmarkers in the selection process is often called genetic marker enhancedselection or marker-assisted selection. Molecular markers may also beused to identify and exclude certain sources of germplasm as parentalvarieties or ancestors of a plant by providing a means of trackinggenetic profiles through crosses.

Particular markers used for these purposes are not limited to the set ofmarkers disclosed herein, but may include any type of marker and markerprofile which provides a means of distinguishing varieties. In additionto being used for identification of lettuce cultivar Regency 3.0, ahybrid produced through the use of Regency 3.0, and the identificationor verification of pedigree for progeny plants produced through the useof Regency 3.0, a genetic marker profile is also useful in developing alocus conversion of Regency 3.0.

Means of performing genetic marker profiles using SNP and SSRpolymorphisms are well known in the art. SNPs are genetic markers basedon a polymorphism in a single nucleotide. A marker system based on SNPscan be highly informative in linkage analysis relative to other markersystems in that multiple alleles may be present.

The SSR profile of lettuce cultivar Regency 3.0 can be used to identifyplants comprising lettuce cultivar Regency 3.0 as a parent, since suchplants will comprise the same homozygous alleles as lettuce cultivarRegency 3.0. Because the lettuce variety is essentially homozygous atall relevant loci, most loci should have only one type of allelepresent. In contrast, a genetic marker profile of an F₁ progeny shouldbe the sum of those parents, e.g., if one parent was homozygous forallele x at a particular locus, and the other parent homozygous forallele y at that locus, then the F₁ progeny will be xy (heterozygous) atthat locus. Subsequent generations of progeny produced by selection andbreeding are expected to be of genotype x (homozygous), y (homozygous),or xy (heterozygous) for that locus position. When the F₁ plant isselfed or sibbed for successive filial generations, the locus should beeither x or y for that position.

In addition, plants and plant parts substantially benefiting from theuse of lettuce cultivar Regency 3.0 in their development, such aslettuce cultivar Regency 3.0 comprising a locus conversion, backcrossconversion, transgene, or genetic sterility factor, may be identified byhaving a molecular marker profile with a high percent identity tolettuce cultivar Regency 3.0. Such a percent identity might be 95%, 96%,97%, 98%, 99%, 99.5%, or 99.9% identical to lettuce cultivar Regency3.0.

The SSR profile of lettuce cultivar Regency 3.0 can also be used toidentify essentially derived varieties and other progeny varietiesdeveloped from the use of lettuce cultivar Regency 3.0, as well as cellsand other plant parts thereof. Such plants may be developed using themarkers identified in WO 00/31964, U.S. Pat. Nos. 6,162,967, and7,288,386. Progeny plants and plant parts produced using lettucecultivar Regency 3.0 may be identified by having a molecular markerprofile of at least 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%,75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%,89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99.5% geneticcontribution from lettuce cultivar Regency 3.0, as measured by eitherpercent identity or percent similarity. Such progeny may be furthercharacterized as being within a pedigree distance of lettuce cultivarRegency 3.0, such as within 1, 2, 3, 4, or 5 or less cross-pollinationsto a lettuce plant other than lettuce cultivar Regency 3.0 or a plantthat has lettuce cultivar Regency 3.0 as a progenitor. Unique molecularprofiles may be identified with other molecular tools such as SNPs andRFLPs.

While determining the genotypic profile of the plants described supra,several unique SSR profiles may also be identified which did not appearin either parent of such plant. Such unique SSR profiles may ariseduring the breeding process from recombination or mutation. Acombination of several unique alleles provides a means of identifying aplant variety, an F₁ progeny produced from such variety, and progenyproduced from such variety.

Molecular data from Regency 3.0 may be used in a plant breeding process.Nucleic acids may be isolated from a seed of Regency 3.0 or from aplant, plant part, or cell produced by growing a seed of Regency 3.0, orfrom a seed of Regency 3.0 with a locus conversion, or from a plant,plant part, or cell of Regency 3.0 with a locus conversion. One or morepolymorphisms may be isolated from the nucleic acids. A plant having oneor more of the identified polymorphisms may be selected and used in aplant breeding method to produce another plant.

Introduction of a New Trait or Locus into Lettuce Cultivar Regency 3.0

Cultivar Regency 3.0 represents a new base genetic variety into which anew locus or trait may be introgressed. Backcrossing and directtransformation represent two important methods that can be used toaccomplish such an introgression.

Single Locus Conversions

When the term “lettuce plant” is used in the context of the presentinvention, this also includes any single locus conversions of thatvariety. The term “single locus converted plant” or “single geneconverted plant” refers to those lettuce plants which are developed bybackcrossing or genetic engineering, wherein essentially all of thedesired morphological and physiological characteristics of a variety arerecovered in addition to the one or more genes transferred into thevariety via the backcrossing technique or genetic engineering.Backcrossing methods can be used with the present invention to improveor introduce a characteristic into the variety.

A backcross conversion of lettuce cultivar Regency 3.0 occurs when DNAsequences are introduced through backcrossing (Hallauer, et al., “CornBreeding,” Corn and Corn Improvements, No. 18, pp. 463-481 (1988)), withlettuce cultivar Regency 3.0 utilized as the recurrent parent. Bothnaturally occurring and transgenic DNA sequences may be introducedthrough backcrossing techniques. A backcross conversion may produce aplant with a trait or locus conversion in at least two or morebackcrosses, including at least 2 crosses, at least 3 crosses, at least4 crosses, at least 5 crosses, and the like. Molecular marker assistedbreeding or selection may be utilized to reduce the number ofbackcrosses necessary to achieve the backcross conversion. For example,see, Openshaw, S. J., et al., Marker-assisted Selection in BackcrossBreeding, Proceedings Symposium of the Analysis of Molecular Data, CropScience Society of America, Corvallis, Oreg. (August 1994), where it isdemonstrated that a backcross conversion can be made in as few as twobackcrosses.

The complexity of the backcross conversion method depends on the type oftrait being transferred (single genes or closely linked genes ascompared to unlinked genes), the level of expression of the trait, thetype of inheritance (cytoplasmic or nuclear), and the types of parentsincluded in the cross. It is understood by those of ordinary skill inthe art that for single gene traits that are relatively easy toclassify, the backcross method is effective and relatively easy tomanage. (See, Hallauer, et al., Corn and Corn Improvement, Sprague andDudley, Third Ed. (1998)). Desired traits that may be transferredthrough backcross conversion include, but are not limited to, sterility(nuclear and cytoplasmic), fertility restoration, nutritionalenhancements, drought tolerance, nitrogen utilization, altered fattyacid profile, modified fatty acid metabolism, modified carbohydratemetabolism, industrial enhancements, yield stability, yield enhancement,disease resistance (bacterial, fungal, or viral), insect resistance, andherbicide resistance. In addition, an introgression site itself, such asan FRT site, Lox site, or other site specific integration site, may beinserted by backcrossing and utilized for direct insertion of one ormore genes of interest into a specific plant variety.

A single locus may contain several transgenes, such as a transgene fordisease resistance that, in the same expression vector, also contains atransgene for herbicide resistance. The gene for herbicide resistancemay be used as a selectable marker and/or as a phenotypic trait. Asingle locus conversion of site specific integration system allows forthe integration of multiple genes at a known recombination site in thegenome. At least one, at least two or at least three and less than ten,less than nine, less than eight, less than seven, less than six, lessthan five or less than four locus conversions may be introduced into theplant by backcrossing, introgression or transformation to express thedesired trait, while the plant, or a plant grown from the seed, plantpart or plant cell, otherwise retains the phenotypic characteristics ofthe deposited seed when grown under the same environmental conditions.

The backcross conversion may result from either the transfer of adominant allele or a recessive allele. Selection of progeny containingthe trait of interest is accomplished by direct selection for a traitassociated with a dominant allele. Transgenes transferred viabackcrossing typically function as a dominant single gene trait and arerelatively easy to classify. Selection of progeny for a trait that istransferred via a recessive allele requires growing and selfing thefirst backcross generation to determine which plants carry the recessivealleles. Recessive traits may require additional progeny testing insuccessive backcross generations to determine the presence of the locusof interest. The last backcross generation is usually selfed to givepure breeding progeny for the gene(s) being transferred, although abackcross conversion with a stably introgressed trait may also bemaintained by further backcrossing to the recurrent parent withselection for the converted trait.

Along with selection for the trait of interest, progeny are selected forthe phenotype of the recurrent parent. The backcross is a form ofinbreeding, and the features of the recurrent parent are automaticallyrecovered after successive backcrosses. Poehlman, Breeding Field Crops,p. 204 (1987). Poehlman suggests from one to four or more backcrosses,but as noted above, the number of backcrosses necessary can be reducedwith the use of molecular markers. Other factors, such as a geneticallysimilar donor parent, may also reduce the number of backcrossesnecessary. As noted by Poehlman, backcrossing is easiest for simplyinherited, dominant, and easily recognized traits.

One process for adding or modifying a trait or locus in lettuce cultivarRegency 3.0 comprises crossing lettuce cultivar Regency 3.0 plants grownfrom lettuce cultivar Regency 3.0 seed with plants of another lettucevariety that comprise the desired trait or locus, selecting F₁ progenyplants that comprise the desired trait or locus to produce selected F₁progeny plants, crossing the selected progeny plants with the lettucecultivar Regency 3.0 plants to produce backcross progeny plants,selecting for backcross progeny plants that have the desired trait orlocus and the morphological characteristics of lettuce cultivar Regency3.0 to produce selected backcross progeny plants, and backcrossing tolettuce cultivar Regency 3.0 three or more times in succession toproduce selected fourth or higher backcross progeny plants that comprisesaid trait or locus. The modified lettuce cultivar Regency 3.0 may befurther characterized as having the physiological and morphologicalcharacteristics of lettuce cultivar Regency 3.0 listed in Table 1 asdetermined at the 5% significance level when grown in the sameenvironmental conditions and/or may be characterized by percentsimilarity or identity to lettuce cultivar Regency 3.0 as determined bySSR markers. The above method may be utilized with fewer backcrosses inappropriate situations, such as when the donor parent is highly relatedor markers are used in the selection step. Desired traits that may beused include those nucleic acids known in the art, some of which arelisted herein, that will affect traits through nucleic acid expressionor inhibition. Desired loci include the introgression of FRT, Lox, andother sites for site specific integration, which may also affect adesired trait if a functional nucleic acid is inserted at theintegration site.

In addition, the above process and other similar processes describedherein may be used to produce first generation progeny lettuce seed byadding a step at the end of the process that comprises crossing lettucecultivar Regency 3.0 with the introgressed trait or locus with adifferent lettuce plant and harvesting the resultant first generationprogeny lettuce seed.

Methods for Genetic Engineering of Lettuce

With the advent of molecular biological techniques that have allowed theisolation and characterization of genes that encode specific proteinproducts, scientists in the field of plant biology developed a stronginterest in engineering the genome of plants (genetic engineering) tocontain and express foreign genes, or additional, or modified versionsof native, or endogenous, genes (perhaps driven by different promoters)in order to alter the traits of a plant in a specific manner. Plantsaltered by genetic engineering are often referred to as ‘geneticallymodified’. Any DNA sequences, whether from a different species or fromthe same species, which are introduced into the genome usingtransformation and/or various breeding methods, are referred to hereincollectively as “transgenes.” Over the last fifteen to twenty years,several methods for producing transgenic plants have been developed, andthe present invention, in particular embodiments, also relates totransformed versions of the claimed cultivar.

Vectors used for the transformation of lettuce cells are not limited solong as the vector can express an inserted DNA in the cells. Forexample, vectors comprising promoters for constitutive gene expressionin lettuce cells (e.g., cauliflower mosaic virus 35S promoter) andpromoters inducible by exogenous stimuli can be used. Examples ofsuitable vectors include pBI binary vector. The “lettuce cell” intowhich the vector is to be introduced includes various forms of lettucecells, such as cultured cell suspensions, protoplasts, leaf sections,and callus. A vector can be introduced into lettuce cells by knownmethods, such as the polyethylene glycol method, polycation method,electroporation, Agrobacterium-mediated transfer, particle bombardmentand direct DNA uptake by protoplasts. See, e.g., Pang et al. (The PlantJ., 9, 899-909, 1996).

Numerous methods for plant transformation have been developed, includingbiological and physical, plant transformation protocols. See, forexample, Miki, et al., “Procedures for Introducing Foreign DNA intoPlants” in Methods in Plant Molecular Biology and Biotechnology, Glickand Thompson (Eds.), CRC Press, Inc., Boca Raton, pp. 67-88 (1993). Inaddition, expression vectors and in vitro culture methods for plant cellor tissue transformation and regeneration of plants are available. See,for example, Gruber, et al., “Vectors for Plant Transformation” inMethods in Plant Molecular Biology and Biotechnology, Glick and Thompson(Eds.), CRC Press, Inc., Boca Raton, pp. 89-119 (1993).

A. Agrobacterium-Mediated Transformation:

One method for introducing an expression vector into plants is based onthe natural transformation system of Agrobacterium. See, for example,Horsch, et al., Science, 227:1229 (1985). A. tumefaciens and A.rhizogenes are plant pathogenic soil bacteria which geneticallytransform plant cells. The Ti and Ri plasmids of A. tumefaciens and A.rhizogenes, respectively, carry genes responsible for genetictransformation of the plant. See, for example, Kado, C. I., Crit. Rev.Plant Sci., 10:1 (1991). Descriptions of Agrobacterium vector systemsand methods for Agrobacterium-mediated gene transfer are provided byGruber, et al., supra, Miki, et al., supra, and Moloney, et al., PlantCell Rep., 8:238 (1989). See also, U.S. Pat. No. 5,563,055 (Townsend andThomas), issued Oct. 8, 1996.

Agrobacterium-mediated transfer is a widely applicable system forintroducing gene loci into plant cells, including lettuce. An advantageof the technique is that DNA can be introduced into whole plant tissues,thereby bypassing the need for regeneration of an intact plant from aprotoplast. Modern Agrobacterium transformation vectors are capable ofreplication in E. coli as well as Agrobacterium, allowing for convenientmanipulations (Klee et al., Bio. Tech., 3(7):637-642, 1985). Moreover,recent technological advances in vectors for Agrobacterium-mediated genetransfer have improved the arrangement of genes and restriction sites inthe vectors to facilitate the construction of vectors capable ofexpressing various polypeptide coding genes. The vectors described haveconvenient multi-linker regions flanked by a promoter and apolyadenylation site for direct expression of inserted polypeptidecoding genes. Additionally, Agrobacterium containing both armed anddisarmed Ti genes can be used for transformation.

In those plant strains where Agrobacterium-mediated transformation isefficient, it is the method of choice because of the facile and definednature of the gene locus transfer. The use of Agrobacterium-mediatedplant integrating vectors to introduce DNA into plant cells is wellknown in the art (Fraley et al., Bio. Tech., 3(7):629-635, 1985; U.S.Pat. No. 5,563,055). For example, U.S. Pat. No. 5,349,124 describes amethod of transforming lettuce plant cells using Agrobacterium-mediatedtransformation. By inserting a chimeric gene having a DNA codingsequence encoding for the full-length B.t. toxin protein that expressesa protein toxic toward Lepidopteran larvae, this methodology resulted inlettuce having resistance to such insects.

B. Direct Gene Transfer:

Several methods of plant transformation, collectively referred to asdirect gene transfer, have been developed as an alternative toAgrobacterium-mediated transformation. A generally applicable method fordelivering transforming DNA segments to plant cells ismicroprojectile-mediated transformation, or microprojectile bombardment.In this method, particles are coated with nucleic acids and deliveredinto cells by a propelling force. Sanford, et al., Part. Sci. Technol.,5:27 (1987); Sanford, J. C., Trends Biotech., 6:299 (1988); Klein, etal., Bio/technology, 6:559-563 (1988); Sanford, J. C., Physiol Plant,7:206 (1990); Klein, et al., Bio/technology, 10:268 (1992). See also,U.S. Pat. No. 5,015,580 (Christou, et al.), issued May 14, 1991; U.S.Pat. No. 5,322,783 (Tomes, et al.), issued Jun. 21, 1994.

Another method for physical delivery of DNA to plants is sonication oftarget cells. Zhang, et al., Bio/technology, 9:996 (1991).Alternatively, liposome and spheroplast fusion have been used tointroduce expression vectors into plants. Deshayes, et al., EMBO 1,4:2731 (1985); Christou, et al., PNAS, 84:3962 (1987). Direct uptake ofDNA into protoplasts using CaCl₂ precipitation, calcium phosphateprecipitation, polyethylene glycol treatment, polyvinyl alcohol, orpoly-L-ornithine has also been reported. See, e.g., Potrykus et al.,Mol. Gen. Genet., 199:183-188, 1985; Omirulleh et al., Plant Mol. Biol21(3):415-428, 1993; Fromm et al., Nature, 312:791-793, 1986; Uchimiyaet al., Mol. Gen. Genet., 204:204, 1986; Marcotte et al., Nature,335:454, 1988; Hain, et al., Mol. Gen. Genet., 199:161, 1985 and Draper,et al., Plant Cell Physiol. 23:451, 1982.

Electroporation of protoplasts and whole cells and tissues has also beendescribed. Donn, et al., In Abstracts of VIIth International Congress onPlant Cell and Tissue Culture IAPTC, A2-38, p. 53, 1990; D'Halluin, etal., Plant Cell, 4:1495-1505, 1992; and Spencer, et al., Plant Mol.Biol., 24:51-61, 1994. Another illustrative embodiment of a method fordelivering DNA into plant cells by acceleration is the BiolisticsParticle Delivery System, which can be used to propel particles coatedwith DNA or cells through a screen, such as a stainless steel or Nytexscreen, onto a surface covered with target lettuce cells.

Transformation of plants and expression of foreign genetic elements isexemplified in Choi et al., Plant Cell Rep., 13: 344-348, 1994 and Ellulet al., Theor. Appl. Genet., 107:462-469, 2003.

Following transformation of lettuce target tissues, expression ofselectable marker genes allows for preferential selection of transformedcells, tissues, and/or plants, using regeneration and selection methodsnow well known in the art.

The methods described herein for transformation would typically be usedfor producing a transgenic variety. The transgenic variety could then becrossed, with another (non-transformed or transformed) variety, in orderto produce a new transgenic variety. Alternatively, a genetic traitwhich has been engineered into a particular lettuce cultivar using thetransformation techniques described could be moved into another cultivarusing traditional backcrossing techniques that are well known in theplant breeding arts. For example, a backcrossing approach could be usedto move an engineered trait from a public, non-elite variety into anelite variety, or from a variety containing a foreign gene in its genomeinto a variety or varieties which do not contain that gene. As usedherein, “crossing” can refer to a simple X by Y cross, or the process ofbackcrossing, depending on the context.

Expression Vectors for Lettuce Transformation: Marker Genes

Expression vectors include at least one genetic marker, operably linkedto a regulatory element (for example, a promoter) that allowstransformed cells containing the marker to be either recovered bynegative selection, i.e., inhibiting growth of cells that do not containthe selectable marker gene, or by positive selection, i.e., screeningfor the product encoded by the genetic marker. Many commonly usedselectable marker genes for plant transformation are well known in thetransformation arts, and include, for example, genes that code forenzymes that metabolically detoxify a selective chemical agent which maybe an antibiotic or an herbicide, or genes that encode an altered targetwhich is insensitive to the inhibitor. A few positive selection methodsare also known in the art.

One commonly used selectable marker gene for plant transformation is theneomycin phosphotransferase II (nptII) gene, isolated from transposonTn5, which when placed under the control of plant regulatory signalsconfers resistance to kanamycin. Fraley, et al., PNAS, 80:4803 (1983).Another commonly used selectable marker gene is the hygromycinphosphotransferase gene which confers resistance to the antibiotichygromycin. Vanden Elzen, et al., Plant Mol. Biol., 5:299 (1985).

Additional selectable marker genes of bacterial origin that conferresistance to antibiotics include gentamycin acetyl transferase,streptomycin phosphotransferase, aminoglycoside-3′-adenyl transferase,the bleomycin resistance determinant. Hayford, et al., Plant Physiol.,86:1216 (1988); Jones, et al., Mol. Gen. Genet., 210:86 (1987); Svab, etal., Plant Mol. Biol., 14:197 (1990); Hille, et al., Plant Mol. Biol.,7:171 (1986). Other selectable marker genes confer resistance toherbicides such as glyphosate, glufosinate, or bromoxynil. Comai, etal., Nature, 317:741-744 (1985); Gordon-Kamm, et al., Plant Cell,2:603-618 (1990); and Stalker, et al., Science, 242:419-423 (1988).

Selectable marker genes for plant transformation that are not ofbacterial origin include, for example, mouse dihydrofolate reductase,plant 5-enolpyruvylshikimate-3-phosphate synthase, and plantacetolactate synthase. Eichholtz, et al., Somatic Cell Mol. Genet.,13:67 (1987); Shah, et al., Science, 233:478 (1986); and Charest, etal., Plant Cell Rep., 8:643 (1990).

Another class of marker genes for plant transformation requiresscreening of presumptively transformed plant cells rather than directgenetic selection of transformed cells for resistance to a toxicsubstance such as an antibiotic. These genes are particularly useful toquantify or visualize the spatial pattern of expression of a gene inspecific tissues and are frequently referred to as reporter genesbecause they can be fused to a gene or gene regulatory sequence for theinvestigation of gene expression. Commonly used genes for screeningpresumptively transformed cells include α-glucuronidase (GUS),α-galactosidase, luciferase and chloramphenicol, acetyltransferase.Jefferson, R. A., Plant Mol. Biol., 5:387 (1987); Teeri, et al., EMBO 1,8:343 (1989); Koncz, et al., PNAS, 84:131 (1987); and DeBlock, et al.,EMBO J., 3:1681 (1984).

In vivo methods for visualizing GUS activity that do not requiredestruction of plant tissues are available. Molecular Probes,Publication 2908, IMAGENE GREEN, pp. 1-4 (1993) and Naleway, et al., J.Cell Biol., 115:151a (1991). However, these in vivo methods forvisualizing GUS activity have not proven useful for recovery oftransformed cells because of low sensitivity, high fluorescentbackgrounds, and limitations associated with the use of luciferase genesas selectable markers.

More recently, a gene encoding Green Fluorescent Protein (GFP) has beenutilized as a marker for gene expression in prokaryotic and eukaryoticcells. Chalfie, et al., Science, 263:802 (1994). GFP and mutants of GFPmay be used as screenable markers.

Expression Vectors for Lettuce Transformation: Promoters

Genes included in expression vectors must be driven by a nucleotidesequence comprising a regulatory element (for example, a promoter).Several types of promoters are now well known in the transformationarts, as are other regulatory elements that can be used alone or incombination with promoters.

As used herein, “promoter” includes reference to a region of DNAupstream from the start of transcription and involved in recognition andbinding of RNA polymerase and other proteins to initiate transcription.A “plant promoter” is a promoter capable of initiating transcription inplant cells. Examples of promoters under developmental control includepromoters that preferentially initiate transcription in certain tissues,such as leaves, roots, seeds, fibers, xylem vessels, tracheids, orsclerenchyma. Such promoters are referred to as “tissue-preferred.”Promoters which initiate transcription only in certain tissue arereferred to as “tissue-specific.” A “cell type” specific promoterprimarily drives expression in certain cell types in one or more organs,for example, vascular cells in roots or leaves. An “inducible” promoteris a promoter which is under environmental control. Examples ofenvironmental conditions that may effect transcription by induciblepromoters include anaerobic conditions or the presence of light.Tissue-specific, tissue-preferred, cell type specific, and induciblepromoters constitute the class of “non-constitutive” promoters. A“constitutive” promoter is a promoter which is active under mostenvironmental conditions.

A. Inducible Promoters:

An inducible promoter is operably linked to a gene for expression inlettuce. Optionally, the inducible promoter is operably linked to anucleotide sequence encoding a signal sequence which is operably linkedto a gene for expression in lettuce. With an inducible promoter, therate of transcription increases in response to an inducing agent.

Any inducible promoter can be used in the instant invention. See Ward,et al., Plant Mol. Biol., 22:361-366 (1993). Exemplary induciblepromoters include, but are not limited to, that from the ACEI systemwhich responds to copper (Meft, et al., PNAS, 90:4567-4571 (1993)); In2gene from maize which responds to benzenesulfonamide herbicide safeners(Hershey, et al., Mol. Gen. Genet., 227:229-237 (1991) and Gatz, et al.,Mol. Gen. Genet., 243:32-38 (1994)) or Tet repressor from Tn10 (Gatz, etal., Mol. Gen. Genet., 227:229-237 (1991)). A particularly preferredinducible promoter is a promoter that responds to an inducing agent towhich plants do not normally respond. An exemplary inducible promoter isthe inducible promoter from a steroid hormone gene, the transcriptionalactivity of which is induced by a glucocorticosteroid hormone. Schena,et al., PNAS, 88:0421 (1991).

B. Constitutive Promoters:

A constitutive promoter is operably linked to a gene for expression inlettuce or the constitutive promoter is operably linked to a nucleotidesequence encoding a signal sequence which is operably linked to a genefor expression in lettuce.

Many different constitutive promoters can be utilized in the instantinvention. Exemplary constitutive promoters include, but are not limitedto, the promoters from plant viruses such as the 35S promoter from CaMV(Odell, et al., Nature, 313:810-812 (1985)) and the promoters from suchgenes as rice actin (McElroy, et al., Plant Cell, 2:163-171 (1990));ubiquitin (Christensen, et al., Plant Mol. Biol., 12:619-632 (1989) andChristensen, et al., Plant Mol. Biol., 18:675-689 (1992)); pEMU (Last,et al., Theor. Appl. Genet., 81:581-588 (1991)); MAS (Velten, et al.,EMBO J., 3:2723-2730 (1984)) and maize H3 histone (Lepetit, et al., Mol.Gen. Genet., 231:276-285 (1992) and Atanassova, et al., Plant J., 2(3):291-300 (1992)). The ALS promoter, Xba1/Ncol fragment 5′ to theBrassica napus ALS3 structural gene (or a nucleotide sequence similarityto said Xba1/Ncol fragment), represents a particularly usefulconstitutive promoter. See PCT Application No. WO 96/30530.

C. Tissue-Specific or Tissue-Preferred Promoters:

A tissue-specific promoter is operably linked to a gene for expressionin lettuce. Optionally, the tissue-specific promoter is operably linkedto a nucleotide sequence encoding a signal sequence which is operablylinked to a gene for expression in lettuce. Plants transformed with agene of interest operably linked to a tissue-specific promoter producethe protein product of the transgene exclusively, or preferentially, ina specific tissue.

Any tissue-specific or tissue-preferred promoter can be utilized in theinstant invention. Exemplary tissue-specific or tissue-preferredpromoters include, but are not limited to, a root-preferred promoter,such as that from the phaseolin gene (Murai, et al., Science, 23:476-482(1983) and Sengupta-Gopalan, et al., PNAS, 82:3320-3324 (1985)); aleaf-specific and light-induced promoter such as that from cab orrubisco (Simpson, et al., EMBO J., 4(11):2723-2729 (1985) and Timko, etal., Nature, 318:579-582 (1985)); an anther-specific promoter such asthat from LAT52 (Twell, et al., Mol. Gen. Genet., 217:240-245 (1989)); apollen-specific promoter such as that from Zm13 (Guerrero, et al., Mol.Gen. Genet., 244:161-168 (1993)) or a microspore-preferred promoter suchas that from apg (Twell, et al., Sex. Plant Reprod., 6:217-224 (1993)).

Signal Sequences for Targeting Proteins to Subcellular Compartments

Transport of protein produced by transgenes to a subcellular compartmentsuch as the chloroplast, vacuole, peroxisome, glyoxysome, cell wall, ormitochondrion, or for secretion into the apoplast, is accomplished bymeans of operably linking the nucleotide sequence encoding a signalsequence to the 5′ and/or 3′ region of a gene encoding the protein ofinterest. Targeting sequences at the 5′ and/or 3′ end of the structuralgene may determine, during protein synthesis and processing, where theencoded protein is ultimately compartmentalized.

The presence of a signal sequence directs a polypeptide to either anintracellular organelle or subcellular compartment or for secretion tothe apoplast. Many signal sequences are known in the art. See, forexample, Becker, et al., Plant Mol. Biol., 20:49 (1992); Close, P. S.,Master's Thesis, Iowa State University (1993); Knox, C., et al.,“Structure and Organization of Two Divergent Alpha-Amylase Genes fromBarley,” Plant Mol. Biol., 9:3-17 (1987); Lerner, et al., PlantPhysiol., 91:124-129 (1989); Fontes, et al., Plant Cell, 3:483-496(1991); Matsuoka, et al., PNAS, 88:834 (1991); Gould, et al., J. Cell.Biol., 108:1657 (1989); Creissen, et al., Plant J., 2:129 (1991);Kalderon, et al., A short amino acid sequence able to specify nuclearlocation, Cell, 39:499-509 (1984); and Steifel, et al., Expression of amaize cell wall hydroxyproline-rich glycoprotein gene in early leaf androot vascular differentiation, Plant Cell, 2:785-793 (1990).

Additional Methods for Genetic Engineering of Lettuce

In general, methods to transform, modify, edit or alter plant endogenousgenomic DNA include altering the plant native DNA sequence or apre-existing transgenic sequence including regulatory elements, codingand non-coding sequences. These methods can be used, for example, totarget nucleic acids to pre-engineered target recognition sequences inthe genome. Such pre-engineered target sequences may be introduced bygenome editing or modification. As an example, a genetically modifiedplant variety is generated using “custom” or engineered endonucleasessuch as meganucleases produced to modify plant genomes (see e.g., WO2009/114321; Gao et al. (2010) Plant Journal 1:176-187). Anothersite-directed engineering method is through the use of zinc fingerdomain recognition coupled with the restriction properties ofrestriction enzyme. See e.g., Urnov, et al., (2010) Nat Rev Genet.11(9):636-46; Shukla, et al., (2009) Nature 459 (7245):437-41. Atranscription activator-like (TAL) effector-DNA modifying enzyme (TALEor TALEN) is also used to engineer changes in plant genome. See e.g.,US20110145940, Cermak et al., (2011) Nucleic Acids Res. 39(12) and Bochet al., (2009), Science 326(5959): 1509-12. Site-specific modificationof plant genomes can also be performed using the bacterial type IICRISPR (clustered regularly interspaced short palindromic repeats)/Cas(CRISPR-associated) system. See e.g., Belhaj et al., (2013), PlantMethods 9: 39; The Cas9/guide RNA-based system allows targeted cleavageof genomic DNA guided by a customizable small noncoding RNA in plants(see e.g., WO 2015026883A1, incorporated herein by reference).

A genetic map can be generated that identifies the approximatechromosomal location of an integrated DNA molecule, for example viaconventional restriction fragment length polymorphisms (RFLP),polymerase chain reaction (PCR) analysis, simple sequence repeats (SSR),and single nucleotide polymorphisms (SNP). For exemplary methodologiesin this regard, see Glick and Thompson, Methods in Plant MolecularBiology and Biotechnology, pp. 269-284 (CRC Press, Boca Raton, 1993).

Wang et al. discuss “Large Scale Identification, Mapping and Genotypingof Single-Nucleotide Polymorphisms in the Human Genome”, Science (1998)280:1077-1082, and similar capabilities are increasingly available forthe lettuce genome. Map information concerning chromosomal location isuseful for proprietary protection of a subject transgenic plant. Ifunauthorized propagation is undertaken and crosses made with othergermplasm, the map of the integration region can be compared to similarmaps for suspect plants to determine if the latter have a commonparentage with the subject plant. Map comparisons could involvehybridizations, RFLP, PCR, SSR, sequencing or combinations thereof, allof which are conventional techniques. SNPs may also be used alone or incombination with other techniques.

Lettuce Cultivar Regency 3.0 Further Comprising a Transgene

Transgenes and transformation methods provide means to engineer thegenome of plants to contain and express heterologous genetic elements,including but not limited to foreign genetic elements, additional copiesof endogenous elements, and/or modified versions of native or endogenousgenetic elements, in order to alter at least one trait of a plant in aspecific manner. Any heterologous DNA sequence(s), whether from adifferent species or from the same species, which are inserted into thegenome using transformation, backcrossing, or other methods known to oneof skill in the art are referred to herein collectively as transgenes.The sequences are heterologous based on sequence source, location ofintegration, operably linked elements, or any combination thereof. Oneor more transgenes of interest can be introduced into lettuce cultivarRegency 3.0. Transgenic variants of lettuce cultivar Regency 3.0 plants,seeds, cells, and parts thereof or derived therefrom are provided.Transgenic variants of Regency 3.0 comprise the physiological andmorphological characteristics of lettuce cultivar Regency 3.0, such aslisted in Table 1 as determined at the 5% significance level when grownin the same environmental conditions, and/or may be characterized oridentified by percent similarity or identity to Regency 3.0 asdetermined by SSR or other molecular markers. In some examples,transgenic variants of lettuce cultivar Regency 3.0 are produced byintroducing at least one transgene of interest into lettuce cultivarRegency 3.0 by transforming Regency 3.0 with a polynucleotide comprisingthe transgene of interest. In other examples, transgenic variants oflettuce cultivar Regency 3.0 are produced by introducing at least onetransgene by introgressing the transgene into lettuce cultivar Regency3.0 by crossing.

In one example, a process for modifying lettuce cultivar Regency 3.0with the addition of a desired trait, said process comprisingtransforming a lettuce plant of cultivar Regency 3.0 with a transgenethat confers a desired trait is provided. Therefore, transgenic Regency3.0 lettuce cells, plants, plant parts, and seeds produced from thisprocess are provided. In some examples one more desired traits mayinclude traits such as sterility (nuclear and cytoplasmic), fertilityrestoration, nutritional enhancements, drought tolerance, nitrogenutilization, altered fatty acid profile, modified fatty acid metabolism,modified carbohydrate metabolism, industrial enhancements, yieldstability, yield enhancement, disease resistance (bacterial, fungal, orviral), insect resistance, and herbicide resistance. The specific genemay be any known in the art or listed herein, including but not limitedto a polynucleotide conferring resistance to an ALS-inhibitor herbicide,imidazolinone, sulfonylurea, protoporphyrinogen oxidase (PPO)inhibitors, hydroxyphenyl pyruvate dioxygenase (HPPD) inhibitors,glyphosate, glufosinate, triazine, 2,4-dichlorophenoxyacetic acid(2,4-D), dicamba, broxynil, metribuzin, or benzonitrile herbicides; apolynucleotide encoding a Bacillus thuringiensis polypeptide, apolynucleotide encoding a phytase, a fatty acid desaturase (e.g., FAD-2,FAD-3), galactinol synthase, a raffinose synthetic enzyme; or apolynucleotide conferring resistance to tipburn, Bremia lactucae, corkyroot, Fusarium oxysporum, lettuce big vein virus, lettuce mosaic virus,lettuce necrotic stunt virus, Nasonovia ribisnigri, Sclerotiniasclerotiorum or other plant pathogens.

Foreign Protein Genes and Agronomic Genes

By means of the present invention, plants can be genetically engineeredto express various phenotypes of agronomic interest. Through thetransformation of lettuce, the expression of genes can be altered toenhance disease resistance, insect resistance, herbicide resistance,agronomic, nutritional quality, and other traits. Transformation canalso be used to insert DNA sequences which control or help controlmale-sterility. DNA sequences native to lettuce, as well as non-nativeDNA sequences, can be transformed into lettuce and used to alter levelsof native or non-native proteins. Various promoters, targetingsequences, enhancing sequences, and other DNA sequences can be insertedinto the genome for the purpose of altering the expression of proteins.Reduction of the activity of specific genes (also known as genesilencing or gene suppression) is desirable for several aspects ofgenetic engineering in plants.

Many techniques for gene silencing are well known to one of skill in theart, including, but not limited to, knock-outs (such as by insertion ofa transposable element such as mu (Vicki Chandler, The Maize Handbook,Ch. 118 (Springer-Verlag 1994)) or other genetic elements such as a FRTand Lox that are used for site specific integrations, antisensetechnology (see, e.g., Sheehy, et al., PNAS USA, 85:8805-8809 (1988);and U.S. Pat. Nos. 5,107,065, 5,453,566, and 5,759,829); co-suppression(e.g., Taylor, Plant Cell, 9:1245 (1997); Jorgensen, Trends Biotech.,8(12):340-344 (1990); Flavell, PNAS USA, 91:3490-3496 (1994); Finnegan,et al., Bio/Technology, 12:883-888 (1994); Neuhuber, et al., Mol. Gen.Genet., 244:230-241 (1994)); RNA interference (Napoli, et al., PlantCell, 2:279-289 (1990); U.S. Pat. No. 5,034,323; Sharp, Genes Dev.,13:139-141 (1999); Zamore, et al., Cell, 101:25-33 (2000); Montgomery,et al., PNAS USA, 95:15502-15507 (1998)), virus-induced gene silencing(Burton, et al., Plant Cell, 12:691-705 (2000); Baulcombe, Curr. Op.Plant Bio., 2:109-113 (1999)); target-RNA-specific ribozymes (Haseloff,et al., Nature, 334: 585-591 (1988)); hairpin structures (Smith, et al.,Nature, 407:319-320 (2000); WO 99/53050; WO 98/53083); MicroRNA(Aukerman & Sakai, Plant Cell, 15:2730-2741 (2003)); ribozymes(Steinecke, et al., EMBO J., 11:1525 (1992); Perriman, et al., AntisenseRes. Dev., 3:253 (1993)); oligonucleotide mediated targeted modification(e.g., WO 03/076574 and WO 99/25853); Zn-finger targeted molecules(e.g., WO 01/52620, WO 03/048345, and WO 00/42219); and other methods orcombinations of the above methods known to those of skill in the art.

Likewise, by means of the present invention, agronomic genes can beexpressed in transformed plants. More particularly, plants can begenetically engineered to express various phenotypes of agronomicinterest. Exemplary nucleotide sequences and/or native loci that conferat least one trait of interest, which optionally may be conferred oraltered by genetic engineering, transformation or introgression of atransformed event include, but are not limited to, those categorizedbelow:

A. Genes that Confer Resistance to Pests or Disease and that Encode:

1. Plant disease resistance genes. Plant defenses are often activated byspecific interaction between the product of a disease resistance gene(R) in the plant and the product of a corresponding avirulence (Avr)gene in the pathogen. A plant line can be transformed with a clonedresistance gene to engineer plants that are resistant to specificpathogen strains. See, for example, Jones, et al., Science, 266:789(1994) (cloning of the tomato Cf-9 gene for resistance to Cladosporiumfulvum); Martin, et al., Science, 262:1432 (1993) (tomato Pto gene forresistance to Pseudomonas syringae pv. tomato encodes a protein kinase);and Mindrinos, et al., Cell, 78:1089 (1994) (Arabidopsis RSP2 gene forresistance to Pseudomonas syringae).

2. A Bacillus thuringiensis protein, a derivative thereof, or asynthetic polypeptide modeled thereon. Non-limiting examples of Bttransgenes being genetically engineered are given in the followingpatents and patent applications, and hereby are incorporated byreference for this purpose: U.S. Pat. Nos. 5,188,960; 5,689,052;5,880,275; 5,986,177; 7,105,332; 7,208,474; WO91/14778; WO99/31248;WO01/12731; WO99/24581; WO97/40162; US2002/0151709; US2003/0177528;US2005/0138685; US/20070245427; US2007/0245428; US2006/0241042;US2008/0020966; US2008/0020968; US2008/0020967; US2008/0172762;US2008/0172762; and US2009/0005306.

3. A lectin. See, for example, the disclosure by Van Damme, et al.,Plant Mol. Biol., 24:25 (1994), who disclose the nucleotide sequences ofseveral Clivia miniata mannose-binding lectin genes.

4. A vitamin-binding protein such as avidin. See PCT Application No. US93/06487, the contents of which are hereby incorporated by reference.The application teaches the use of avidin and avidin homologues aslarvicides against insect pests.

5. An enzyme inhibitor, for example, a protease or proteinase inhibitor,or an amylase inhibitor. See, for example, Abe, et al., J. Biol. Chem.,262:16793 (1987) (nucleotide sequence of rice cysteine proteinaseinhibitor); Huub, et al., Plant Mol. Biol., 21:985 (1993) (nucleotidesequence of cDNA encoding tobacco proteinase inhibitor I); and Sumitani,et al., Biosci. Biotech. Biochem., 57:1243 (1993) (nucleotide sequenceof Streptomyces nitrosporeus α-amylase inhibitor); and U.S. Pat. No.5,494,813 (Hepher and Atkinson, issued Feb. 27, 1996).

6. An insect-specific hormone or pheromone, such as an ecdysteroid andjuvenile hormone, a variant thereof, a mimetic based thereon, or anantagonist or agonist thereof. See, for example, the disclosure byHammock, et al., Nature, 344:458 (1990), of baculovirus expression ofcloned juvenile hormone esterase, an inactivator of juvenile hormone.

7. An insect-specific peptide or neuropeptide which, upon expression,disrupts the physiology of the affected pest. For example, see thedisclosures of Regan, J. Biol. Chem., 269:9 (1994) (expression cloningyields DNA coding for insect diuretic hormone receptor); Pratt, et al.,Biochem. Biophys. Res. Comm., 163:1243 (1989) (an allostatin isidentified in Diploptera puntata); Chattopadhyay, et al., CriticalReviews in Microbiology, 30(1):33-54 (2004); Zjawiony, J Nat Prod,67(2):300-310 (2004); Carlini & Grossi-de-Sa, Toxicon, 40(11):1515-1539(2002); Ussuf, et al., Curr Sci., 80(7):847-853 (2001); Vasconcelos &Oliveira, Toxicon, 44(4):385-403 (2004). See also, U.S. Pat. No.5,266,317 to Tomalski, et al., which discloses genes encodinginsect-specific, paralytic neurotoxins.

8. An insect-specific venom produced in nature, by a snake, a wasp, etc.For example, see Pang, et al., Gene, 116:165 (1992), for disclosure ofheterologous expression in plants of a gene coding for a scorpioninsectotoxic peptide.

9. An enzyme responsible for a hyper-accumulation of a monoterpene, asesquiterpene, a steroid, hydroxamic acid, a phenylpropanoid derivative,or another non-protein molecule with insecticidal activity.

10. An enzyme involved in the modification, including thepost-translational modification, of a biologically active molecule; forexample, a glycolytic enzyme, a proteolytic enzyme, a lipolytic enzyme,a nuclease, a cyclase, a transaminase, an esterase, a hydrolase, aphosphatase, a kinase, a phosphorylase, a polymerase, an elastase, achitinase, and a glucanase, whether natural or synthetic. See PCTApplication No. WO 93/02197 in the name of Scott, et al., whichdiscloses the nucleotide sequence of a callase gene. DNA molecules whichcontain chitinase-encoding sequences can be obtained, for example, fromthe ATCC under Accession Nos. 39637 and 67152. See also, Kramer, et al.,Insect Biochem. Mol. Biol., 23:691 (1993), who teach the nucleotidesequence of a cDNA encoding tobacco hornworm chitinase, and Kawalleck,et al., Plant Mol. Biol., 21:673 (1993), who provide the nucleotidesequence of the parsley ubi4-2 polyubiquitin gene.

11. A molecule that stimulates signal transduction. For example, see thedisclosure by Botella, et al., Plant Mol. Biol., 24:757 (1994), ofnucleotide sequences for mung bean calmodulin cDNA clones, and Griess,et al., Plant Physiol., 104:1467 (1994), who provide the nucleotidesequence of a maize calmodulin cDNA clone.

12. A hydrophobic moment peptide. See PCT Application No. WO 95/16776(disclosure of peptide derivatives of tachyplesin which inhibit fungalplant pathogens) and PCT Application No. WO 95/18855 (teaches syntheticantimicrobial peptides that confer disease resistance), the respectivecontents of which are hereby incorporated by reference.

13. A membrane permease, a channel former, or a channel blocker. Forexample, see the disclosure of Jaynes, et al., Plant Sci., 89:43 (1993),of heterologous expression of a cecropin-β, lytic peptide analog torender transgenic tobacco plants resistant to Pseudomonas solanacearum.

14. A viral-invasive protein or a complex toxin derived therefrom. Forexample, the accumulation of viral coat proteins in transformed plantcells imparts resistance to viral infection and/or disease developmenteffected by the virus from which the coat protein gene is derived, aswell as by related viruses. See Beachy, et al., Ann. Rev. Phytopathol.,28:451 (1990). Coat protein-mediated resistance has been conferred upontransformed plants against alfalfa mosaic virus, cucumber mosaic virus,tobacco streak virus, potato virus X, potato virus Y, tobacco etchvirus, tobacco rattle virus, and tobacco mosaic virus. Id.

15. An insect-specific antibody or an immunotoxin derived therefrom.Thus, an antibody targeted to a critical metabolic function in theinsect gut would inactivate an affected enzyme, killing the insect. SeeTaylor, et al., Abstract #497, Seventh Int'l Symposium on MolecularPlant-Microbe Interactions, Edinburgh, Scotland (1994) (enzymaticinactivation in transgenic tobacco via production of single-chainantibody fragments).

16. A virus-specific or pathogen protein specific antibody. See, forexample, Safarnejad, et al. (2011) Biotechnology Advances 29(6):961-971, reviewing antibody-mediated resistance against plant pathogens.

17. A developmental-arrestive protein produced in nature by a pathogenor a parasite. Thus, fungal endo-α-1, 4-D-polygalacturonases facilitatefungal colonization and plant nutrient released by solubilizing plantcell wall homo-α-1,4-D-galacturonase. See Lamb, et al., Bio/technology,10:1436 (1992). The cloning and characterization of a gene which encodesa bean endopolygalacturonase-inhibiting protein is described by Toubart,et al., Plant 1, 2:367 (1992).

18. A developmental-arrestive protein produced in nature by a plant. Forexample, Logemann, et al., Bio/technology, 10:305 (1992), have shownthat transgenic plants expressing the barley ribosome-inactivating genehave an increased resistance to fungal disease.

19. Genes involved in the Systemic Acquired Resistance (SAR) Responseand/or the pathogenesis-related genes. See Fu et al. (2013) Annu RevPlant Biol. 64:839-863, Luna et al. (2012) Plant Physiol. 158:844-853,Pieterse & Van Loon (2004) Curr Opin Plant Bio 7:456-64; and Somssich(2003) Cell 113:815-816.

20. Antifungal genes. See, Ceasar et al. (2012) Biotechnol Lett34:995-1002; Bushnell et al. (1998) Can J Plant Path 20:137-149. Also,see US Patent Application Publications US2002/0166141; US2007/0274972;US2007/0192899; US2008/0022426; and U.S. Pat. Nos. 6,891,085; 7,306,946;and 7,598,346.

21. Detoxification genes, such as for fumonisin, beauvericin,moniliformin, and zearalenone and their structurally-relatedderivatives. For example, see Schweiger et al. (2013) Mol Plant MicrobeInteract. 26:781-792 and U.S. Pat. Nos. 5,716,820; 5,792,931; 5,798,255;5,846,812; 6,083,736; 6,538,177; 6,388,171; and 6,812,380.

22. Cystatin and cysteine proteinase inhibitors. See, for example,Popovic et al. (2013) Phytochemistry 94:53-59. van der Linde et al.(2012) Plant Cell 24:1285-1300 and U.S. Pat. No. 7,205,453.

23. Defensin genes. See, for example, De Coninck et al. (2013) FungalBiology Reviews 26: 109-120, International Patent PublicationWO03/000863 and U.S. Pat. Nos. 6,911,577; 6,855,865; 6,777,592; and7,238,781.

24. A lettuce mosaic potyvirus (LMV) coat protein gene introduced intoLatuca sativa in order to increase its resistance to LMV infection. SeeDinant, et al., Mol. Breeding, 3:1, 75-86 (1997).

Any of the above listed disease or pest resistance genes (1-24) can beintroduced into the claimed lettuce cultivar through a variety of meansincluding but not limited to transformation and crossing.

B. Genes that Confer Resistance to an Herbicide:

1. An herbicide that inhibits the growing point or meristem, such as animidazolinone or a sulfonylurea. Exemplary genes in this category codefor mutant ALS and AHAS enzyme as described, for example, by Lee, etal., EMBO 1, 7:1241 (1988) and Miki, et al., Theor. Appl. Genet., 80:449(1990), respectively. See also, U.S. Pat. Nos. 5,084,082; 5,605,011;5,013,659; 5,141,870; 5,767,361; 5,731,180; 5,304,732; 4,761,373;5,331,107; 5,928,937; and 5,378,824; US2007/0214515; US2013/0254944; andWO96/33270.

2. Glyphosate (resistance conferred by mutant5-enolpyruvlshikimate-3-phosphate synthase (EPSPS) and aroA genes,respectively) and other phosphono compounds, such as glufosinate(phosphinothricin acetyl transferase (PAT) and Streptomyceshygroscopicus phosphinothricin-acetyl transferase PAT bar genes), andpyridinoxy or phenoxy proprionic acids and cyclohexones (ACCaseinhibitor-encoding genes). See, for example, U.S. Pat. No. 4,940,835 toShah, et al., which discloses the nucleotide sequence of a form of EPSPSwhich can confer glyphosate resistance. In addition, glyphosateresistance can be imparted to plants by the over expression of genesencoding glyphosate N-acetyltransferase. See, for example,US2004/0082770; US2005/0246798; and US2008/0234130 which areincorporated herein by reference for this purpose. A DNA moleculeencoding a mutant aroA gene can be obtained under ATCC Accession No.39256, and the nucleotide sequence of the mutant gene is disclosed inU.S. Pat. No. 4,769,061 to Comai. See also, Umaballava-Mobapathie inTransgenic Research, 8:1, 33-44 (1999) that discloses Latuca sativaresistant to glufosinate. European Patent Application No. 0 333 033 toKumada, et al., and U.S. Pat. No. 4,975,374 to Goodman, et al., disclosenucleotide sequences of glutamine synthetase genes which conferresistance to herbicides, such as L-phosphinothricin. The nucleotidesequence of a phosphinothricin-acetyl-transferase gene is provided inEuropean Application No. 0 242 246 to Leemans, et al. DeGreef, et al.,Bio/technology, 7:61 (1989), describe the production of transgenicplants that express chimeric bar genes coding for phosphinothricinacetyl transferase activity. Exemplary of genes conferring resistance tophenoxy proprionic acids and cyclohexones, such as sethoxydim andhaloxyfop, are the Acc1-S1, Acc1-S2, and Acc1-S3 genes described byMarshall, et al., Theor. Appl. Genet., 83:435 (1992). For otherpolynucleotides and/or methods or uses see also U.S. Pat. Nos.6,566,587; 6,338,961; 6,248,876; 6,040,497; 5,804,425; 5,633,435;5,145,783; 4,971,908; 5,312,910; 5,188,642; 4,940,835; 5,866,775;6,225,114; 6,130,366; 5,310,667; 4,535,060; 4,769,061; 5,633,448;5,510,471; RE 36,449; RE 37,287; 7,608,761; 7,632,985; 8,053,184;6,376,754; 7,407,913; and 5,491,288; EP1173580; WO01/66704; EP1173581;US2012/0070839; US2005/0223425; US2007/0197947; US2010/0100980;US2011/0067134; and EP1173582, which are incorporated herein byreference for this purpose.

3. An herbicide that inhibits photosynthesis, such as a triazine (psbAand gs+ genes) and a benzonitrile (nitrilase gene). Przibilla, et al.,Plant Cell, 3:169 (1991), describe the transformation of Chlamydomonaswith plasmids encoding mutant psbA genes. Nucleotide sequences fornitrilase genes are disclosed in U.S. Pat. No. 4,810,648 to Stalker, andDNA molecules containing these genes are available under ATCC AccessionNos. 53435, 67441, and 67442. Cloning and expression of DNA coding for aglutathione S-transferase is described by Hayes, et al., Biochem. J,285:173 (1992). The herbicide methyl viologen inhibits CO.sub.2assimilation. Foyer et al. (Plant Physiol., 109:1047-1057, 1995)describe a plant overexpressing glutathione reductase (GR) which isresistant to methyl viologen treatment.

4. Acetohydroxy acid synthase, which has been found to make plants thatexpress this enzyme resistant to multiple types of herbicides, has beenintroduced into a variety of plants. See Hattori, et al., Mol. Gen.Genet., 246:419 (1995). Other genes that confer tolerance to herbicidesinclude a gene encoding a chimeric protein of rat cytochrome P4507A1 andyeast NADPH-cytochrome P450 oxidoreductase (Shiota, et al., PlantPhysiol., 106:17 (1994)), genes for glutathione reductase and superoxidedismutase (Aono, et al., Plant Cell Physiol., 36:1687 (1995)), and genesfor various phosphotransferases (Datta, et al., Plant Mol. Biol., 20:619(1992)).

5. Protoporphyrinogen oxidase (PPO; protox) is the target of thePPO-inhibitor class of herbicides; a PPO-inhibitor resistant PPO genewas recently identified in Amaranthus tuberculatus (Patzoldt et al.,PNAS, 103(33):12329-2334, 2006). PPO is necessary for the production ofchlorophyll, which is necessary for all plant survival. The protoxenzyme serves as the target for a variety of herbicidal compounds. Theseherbicides also inhibit growth of all the different species of plantspresent, causing their total destruction. The development of plantscontaining altered protox activity which are resistant to theseherbicides are described in U.S. Pat. Nos. 6,288,306, 6,282,837,5,767,373, and International Publication WO 01/12825.

6. Genes that confer resistance to auxin or synthetic auxin herbicides.For example an aryloxyalkanoate dioxygenase (AAD) gene may conferresistance to arlyoxyalkanoate herbicides, such as 2,4-D, as well aspyridyloxyacetate herbicides, such as described in U.S. Pat. No.8,283,522, and US2013/0035233. In other examples, a dicambamonooxygenase (DMO) is used to confer resistance to dicamba. Otherpolynucleotides of interest related to auxin herbicides and/or usesthereof include, for example, the descriptions found in U.S. Pat. Nos.8,119,380; 7,812,224; 7,884,262; 7,855,326; 7,939,721; 7,105,724;7,022,896; 8,207,092; US2011/067134; and US2010/0279866. Any of theabove listed herbicide genes (1-6) can be introduced into the claimedlettuce cultivar through a variety of means including, but not limitedto, transformation and crossing.

C. Genes that Confer or Contribute to a Value-Added Trait, Such as:

1. Increased iron content of the lettuce, for example, by introducinginto a plant a soybean ferritin gene as described in Goto, et al., ActaHorticulturae., 521, 101-109 (2000).

2. Decreased nitrate content of leaves, for example, by introducing intoa lettuce a gene coding for a nitrate reductase. See, for example,Curtis, et al., Plant Cell Rep., 18:11, 889-896 (1999).

3. Increased sweetness of the lettuce by introducing a gene coding formonellin that elicits a flavor 100,000 times sweeter than sugar on amolar basis. See Penarrubia, et al., Biotechnology, 10:561-564 (1992).

4. Modified fatty acid metabolism, for example, by introducing into aplant an antisense gene of stearyl-ACP desaturase to increase stearicacid content of the plant. See Knultzon, et al., PNAS, 89:2625 (1992).

5. Modified carbohydrate composition effected, for example, byintroducing into plants a gene coding for an enzyme that alters thebranching pattern of starch. See Shiroza, et al., J. Bacteriol., 170:810(1988) (nucleotide sequence of Streptococcus mutantsfructosyltransferase gene); Steinmetz, et al., Mol. Gen. Genet., 20:220(1985) (nucleotide sequence of Bacillus subtilis levansucrase gene);Pen, et al., Bio/technology, 10:292 (1992) (production of transgenicplants that express Bacillus lichenifonnis α-amylase); Elliot, et al.,Plant Mol. Biol., 21:515 (1993) (nucleotide sequences of tomatoinvertase genes); Søgaard, et al., J. Biol. Chem., 268:22480 (1993)(site-directed mutagenesis of barley α-amylase gene); and Fisher, etal., Plant Physiol., 102:1045 (1993) (maize endosperm starch branchingenzyme II).

6. Altered antioxidant content or composition, such as alteration oftocopherol or tocotrienols. See, for example, U.S. Pat. Nos. 6,787,683,7,154,029, WO 00/68393 (involving the manipulation of antioxidant levelsthrough alteration of a phytl prenyl transferase (ppt)); WO 03/082899(through alteration of a homogentisate geranyl geranyl transferase(hggt)). D. Genes that Control Male-Sterility:

1. Introduction of a deacetylase gene under the control of atapetum-specific promoter and with the application of the chemicalN-Ac-PPT. See International Publication WO 01/29237.

2. Introduction of various stamen-specific promoters. See InternationalPublications WO 92/13956 and WO 92/13957.

3. Introduction of the barnase and the barstar genes. See Paul, et al.,Plant Mol. Biol., 19:611-622 (1992).

For additional examples of nuclear male and female sterility systems andgenes, see also, U.S. Pat. Nos. 5,859,341, 6,297,426, 5,478,369,5,824,524, 5,850,014, and 6,265,640, all of which are herebyincorporated by reference.

E. Genes that Affect Abiotic Stress Resistance:

Genes that affect abiotic stress resistance (including but not limitedto flowering, seed development, enhancement of nitrogen utilizationefficiency, altered nitrogen responsiveness, drought resistance ortolerance, cold resistance or tolerance, high or low light intensity,and salt resistance or tolerance) and increased yield under stress. Forexample, see: WO 00/73475 where water use efficiency is altered throughalteration of malate; U.S. Pat. Nos. 5,892,009, 5,965,705, 5,929,305,5,891,859, 6,417,428, 6,664,446, 6,706,866, 6,717,034, 6,801,104, WO2000/060089, WO 2001/026459, WO 2001/035725, WO 2001/034726, WO2001/035727, WO 2001/036444, WO 2001/036597, WO 2001/036598, WO2002/015675, WO 2002/017430, WO 2002/077185, WO 2002/079403, WO2003/013227, WO 2003/013228, WO 2003/014327, WO 2004/031349, WO2004/076638, WO 98/09521, and WO 99/38977 describing genes, includingCBF genes and transcription factors effective in mitigating the negativeeffects of freezing, high salinity, and drought on plants, as well asconferring other positive effects on plant phenotype; U.S. Publ. No.2004/0148654 and WO 01/36596, where abscisic acid is altered in plantsresulting in improved plant phenotype, such as increased yield and/orincreased tolerance to abiotic stress; WO 2000/006341, WO 04/090143,U.S. Pat. Nos. 7,531,723 and 6,992,237, where cytokinin expression ismodified resulting in plants with increased stress tolerance, such asdrought tolerance, and/or increased yield. See also, WO 02/02776, WO2003/052063, JP 2002281975, U.S. Pat. No. 6,084,153, WO 01/64898, andU.S. Pat. Nos. 6,177,275 and 6,107,547 (enhancement of nitrogenutilization and altered nitrogen responsiveness). For ethylenealteration, see, U.S. Publ. Nos. 2004/0128719, 2003/0166197, and WO2000/32761. For plant transcription factors or transcriptionalregulators of abiotic stress, see, e.g., U.S. Publ. Nos. 2004/0098764 or2004/0078852.

Other genes and transcription factors that affect plant growth andagronomic traits, such as yield, flowering, plant growth, and/or plantstructure, can be introduced or introgressed into plants. See, e.g., WO97/49811 (LHY), WO 98/56918 (ESD4), WO 97/10339, U.S. Pat. No. 6,573,430(TFL), U.S. Pat. No. 6,713,663 (FT), U.S. Pat. Nos. 6,794,560, 6,307,126(GAI), WO 96/14414 (CON), WO 96/38560, WO 01/21822 (VRN1), WO 00/44918(VRN2), WO 99/49064 (GI), WO 00/46358 (FRI), WO 97/29123, WO 99/09174(D8 and Rht), WO 2004/076638, and WO 004/031349 (transcription factors).

Tissue Culture

Further reproduction of the variety can occur by tissue culture andregeneration. Tissue culture of various tissues of lettuce andregeneration of plants therefrom is well known and widely published. Forexample, reference may be had to Teng, et al., HortScience, 27:9,1030-1032 (1992); Teng, et al., HortScience, 28:6, 669-1671 (1993);Zhang, et al., Journal of Genetics and Breeding, 46:3, 287-290 (1992);Webb, et al., Plant Cell Tissue and Organ Culture, 38:1, 77-79 (1994);Curtis, et al., Journal of Experimental Botany, 45:279, 1441-1449(1994); Nagata, et al., Journal for the American Society forHorticultural Science, 125:6, 669-672 (2000); and Ibrahim, et al., PlantCell Tissue and Organ Culture, 28(2), 139-145 (1992). It is clear fromthe literature that the state of the art is such that these methods ofobtaining plants are routinely used and have a very high rate ofsuccess. Thus, another aspect of this invention is to provide cellswhich upon growth and differentiation produce lettuce plants having thephysiological and morphological characteristics of variety Regency 3.0.

As used herein, the term “tissue culture” indicates a compositioncomprising isolated cells of the same or a different type or acollection of such cells organized into parts of a plant. Exemplarytypes of tissue cultures are protoplasts, calli, meristematic cells, andplant cells that can generate tissue culture that are intact in plantsor parts of plants, such as leaves, pollen, embryos, roots, root tips,anthers, pistils, flowers, seeds, petioles, suckers, and the like. Meansfor preparing and maintaining plant tissue culture are well known in theart. By way of example, a tissue culture comprising organs has been usedto produce regenerated plants. U.S. Pat. Nos. 5,959,185, 5,973,234, and5,977,445 describe certain techniques, the disclosures of which areincorporated herein by reference.

The present invention further provides a method of producing lettucecomprising obtaining a plant of lettuce cultivar Regency 3.0, whereinthe plant has been cultivated to maturity, and collecting the lettucefrom the plant.

Table

Table 2 shows a comparison of characteristics of lettuce cultivarRegency 3.0 versus similar lettuce cultivar Regency 2.0. Column 1 showsthe characteristic, column 2 shows the results for Regency 3.0, andcolumn 3 shows the results for Regency 2.0.

TABLE 2 Characteristic Regency 3.0 Regency 2.0 Resistance to ResistantResistant lettuce down mildew (California 1-9 (California 1-8) (Bremialactucae) and novels) Seed color Black White

As used herein, the term “plant” includes plant cells, plantprotoplasts, plant cell tissue cultures from which lettuce plants can beregenerated, plant calli, plant clumps, and plant cells that are intactin plants or parts of plants, such as leaves, pollen, embryos,cotyledons, hypocotyl, roots, root tips, anthers, pistils, flowers,ovules, seeds, stems, and the like.

The use of the terms “a,” “an,” and “the,” and similar referents in thecontext of describing the invention (especially in the context of thefollowing claims) are to be construed to cover both the singular and theplural, unless otherwise indicated herein or clearly contradicted bycontext. The terms “comprising,” “having,” “including,” and “containing”are to be construed as open-ended terms (i.e., meaning “including, butnot limited to,”) unless otherwise noted. Recitation of ranges of valuesherein are merely intended to serve as a shorthand method of referringindividually to each separate value falling within the range, unlessotherwise indicated herein, and each separate value is incorporated intothe specification as if it were individually recited herein. Forexample, if the range 10-15 is disclosed, then 11, 12, 13, and 14 arealso disclosed. All methods described herein can be performed in anysuitable order unless otherwise indicated herein or otherwise clearlycontradicted by context. The use of any and all examples, or exemplarylanguage (e.g., “such as”) provided herein, is intended merely to betterilluminate the invention and does not pose a limitation on the scope ofthe invention unless otherwise claimed. No language in the specificationshould be construed as indicating any non-claimed element as essentialto the practice of the invention.

DEPOSIT INFORMATION

A deposit of the Vanguard Seed, Inc. proprietary Lettuce CultivarRegency 3.0 disclosed above and recited in the appended claims has beenmade with the American Type Culture Collection (ATCC), 10801 UniversityBoulevard, Manassas, Va. 20110 under the terms of the Budapest Treaty.The date of deposit was May 22, 2019. The deposit of 25 packets of 25seeds in each packet was taken from the same deposit maintained byVanguard Seed, Inc. since prior to the filing date of this application.All restrictions will be irrevocably removed upon granting of a patent,and the deposit is intended to meet all of the requirements of 37 C.F.R.§§ 1.801-1.809. The ATCC Accession Number is PTA-125932. The depositwill be maintained in the depository for a period of thirty years, orfive years after the last request, or for the enforceable life of thepatent, whichever is longer, and will be replaced as necessary duringthat period.

While a number of exemplary aspects and embodiments have been discussedabove, those of skill in the art will recognize certain modifications,permutations, additions, and sub-combinations thereof. It is thereforeintended that the following appended claims and claims hereafterintroduced are interpreted to include all such modifications,permutations, additions, and sub-combinations as are within their truespirit and scope.

What is claimed is:
 1. A plant, plant part or seed of lettuce cultivarRegency 3.0, wherein a representative sample of seed of said cultivarwas deposited under ATCC Accession No. PTA-125932.
 2. The plant part ofclaim 1, further defined as pollen, a meristem, a cell, or an ovule. 3.A lettuce plant, or a plant part thereof, having all of themorphological and physiological characteristics of the plant of claim 1.4. A tissue or cell culture produced from protoplasts or cells from theplant of claim 1, wherein said cells or protoplasts are produced from aplant part selected from the group consisting of leaf, pollen, embryo,cotyledon, hypocotyl, meristem, root, root tip, pistil, anther, ovule,flower, shoot, stem, seed, stalk and petiole.
 5. A lettuce plantregenerated from the tissue or cell culture of claim 4, wherein theregenerated plant has all of the morphological and physiologicalcharacteristics of cultivar Regency 3.0.
 6. A method of producing alettuce seed, wherein the method comprises crossing the plant of claim 1with itself or a second lettuce plant.
 7. The method of claim 6, whereinthe method comprises crossing the plant of lettuce cultivar Regency 3.0with a second, distinct lettuce plant.
 8. An F₁ lettuce seed produced bythe method of claim
 7. 9. A lettuce plant produced by growing the seedof claim
 8. 10. A plant or seed of lettuce cultivar Regency 3.0 furthercomprising a single locus conversion, wherein a representative sample ofseed of said cultivar was deposited under ATCC Accession No. PTA-125932.11. The plant or seed of claim 10, wherein the single locus conversioncomprises a transgene.
 12. The plant or seed of claim 10, wherein thesingle locus confers a trait selected from the group consisting of malesterility, herbicide resistance, insect resistance, pest resistance,disease resistance, modified fatty acid metabolism, modified seed yield,modified bolting, abiotic stress resistance, a value-added trait,altered seed amino acid composition, site-specific geneticrecombination, and modified carbohydrate metabolism.
 13. The plant orseed of claim 12, wherein the single locus confers resistance to anherbicide selected from the group consisting of glyphosate,sulfonylurea, imidazolinone, dicamba, glufosinate, phenoxy propionicacid, L-phosphinothricin, PPO inhibitors, 2,4-dichlorophenoxyaceticacid, hydroxyphenyl-pyruvate dioxygenase (HPPD) inhibitors, cyclohexone,cyclohexanedione, triazine, benzonitrile, and bromoxynil.
 14. The plantor seed of claim 12, wherein the single locus comprises a transgene. 15.A method for producing a seed of a cultivar Regency 3.0-derived lettuceplant comprising the steps of: (a) crossing the lettuce plant of claim 1with a second lettuce plant; and (b) allowing seed of a Regency3.0-derived lettuce plant to form.
 16. The method of claim 15, whereinthe method further comprises the steps of: (c) crossing a plant grownfrom said Regency 3.0-derived lettuce seed with itself or a secondlettuce plant to yield additional Regency 3.0-derived lettuce seed; (d)growing said additional Regency 3.0-derived lettuce seed of step (c) toyield additional Regency 3.0-derived lettuce plants; and (e) repeatingthe crossing and growing steps of (c) and (d) to generate furtherRegency 3.0-derived lettuce plants.
 17. A method of vegetativelypropagating a plant of lettuce cultivar Regency 3.0, wherein the methodcomprises: (a) collecting a plant part capable of being propagated froma plant of lettuce cultivar Regency 3.0, wherein a representative sampleof seed of said cultivar was deposited under ATCC Accession No.PTA-125932; and (b) producing at least a first rooted plantlet or plantfrom said plant part.
 18. A lettuce plantlet or plant produced by themethod of claim 17, wherein the lettuce plantlet or plant has all of thephysiological and morphological characteristics of lettuce cultivarRegency 3.0.
 19. A method of producing a genetically modified lettuceplant, wherein the method comprises mutation, transformation, geneconversion, genome editing, RNA interference or gene silencing of theplant of claim
 1. 20. A genetically modified lettuce plant produced bythe method of claim 19, wherein said plant is produced bytransformation, gene conversion, genome editing, RNA interference, orgene silencing and otherwise comprises all of the physiological andmorphological characteristics of lettuce cultivar Regency 3.0.
 21. Amethod of determining a genotype of lettuce cultivar Regency 3.0, or afirst generation progeny thereof, the method comprising: (a) obtaining asample of nucleic acids from the plant of claim 1; and (b) detecting apolymorphism in the nucleic acid sample.
 22. A method of producing acommodity plant product, comprising obtaining the plant of claim 1, or aplant part thereof, and producing the commodity plant product from saidplant or plant part thereof, wherein said commodity plant product isselected from the group consisting of fresh lettuce leaf, fresh lettucehead, cut, sliced, ground, pureed, dried, canned, jarred, washed,packaged, frozen and heated leaves.
 23. A commodity plant productproduced by the method of claim 22, wherein the commodity plant productcomprises at least one cell of lettuce cultivar Regency 3.0.